Cell-targeted cytotoxic constructs

ABSTRACT

The present invention is directed generally to cell-targeted cytotoxic constructs comprising a targeting polypeptide, a linking polypeptide and a cytotoxic polypeptide. Preferably, (a) the targeting polypeptide is a R-spondin1 (RSPO1), R-spondin2 (RSPO2) or yoked chorionic gonadotropin (YCG), the linking polypeptide comprises LPXT (SEQ ID NO: 56) or NPXT (SEQ ID NO: 60) as well as others, where X is any amino acid, the linking polypeptide being positioned between the targeting ligand and (c) the cytotoxic moiety is an auristatin or a truncated serine protease, the serine protease having an IIGG (SEQ ID NO: 91), IVGG (SEQ ID NO: 92) or ILGG (SEQ ID NO: 93) at its N-terminus. Such constructs can be used in methods for targeted cell killing such as for treatment cell of proliferative diseases (e.g., cancer).

The present application claims the priority benefit of U.S. ProvisionalApplication Ser. No. 62/295,636, filed Feb. 16, 2016, the entirecontents of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of molecularbiology and recombinant protein production. More particularly, itconcerns the linkage of cytotoxic agents, such as serine proteasepolypeptides (e.g., granzymes) to cell-targeting moieties.

2. Description of Related Art

The successful development of targeted therapeutics (e.g., for cancerapplications) depends on the identification of ligands and antigensspecific for target cells, generation of molecules capable of targetingthose components specifically and, finally, use of highly toxicmolecules for killing of target cells. Immunoconjugates composed ofantibodies and small, toxic drugs or radioisotopes have beensuccessfully tested in vitro, in animal models and have demonstratedactivity in the clinical setting. In addition to the use of smallmolecules for the toxin component, a number of highly cytotoxic proteincomponents, such as diphtheria toxin, ricin A-chain, Pseudomonasexotoxin, and gelonin (rGel), have been used for targeted therapies.However, problems such as capillary leak syndrome, immunogenicity andinadvertent toxicity (to non-targeted cells) continue to limitimplementation of successful therapy, especially for long-term orchronic applications. Thus, there remains a need for highly specific andhighly active toxin molecules and cell-targeting constructs comprisingsuch molecules.

SUMMARY OF THE INVENTION

Certain embodiments of the present disclosure concern compositionsproduced using a sortase reaction. In one embodiment, there is provideda compound comprising a cytotoxic agent conjugated to a sortaserecognition sequence.

In some aspects, the cytotoxic agent is a cytotoxic polypeptide, such asa serine protease. Thus, in some aspects, the present disclosureconcerns sortase serine protease polypeptides and fusion proteinscomprising such sortase serine proteases. In some aspects, a sortaseserine protease polypeptide is conjugated to, or fused with, a celltargeting moiety to provide a cell-targeted cytotoxic construct. Suchconstructs can be used, for example, in the treatment of cellproliferative diseases, such as cancer.

In certain aspects, the serine protease is granzyme B, granzyme A,granzyme H, granzyme K, granzyme M, Cathepsin G, Chymase, Myeloblastin,Kallikrein-14, Complement factor D, PRSS3 protein, Trypsin-1, Serineprotease 57 or PRSSL1 protein. In particular aspects, the serineprotease is Granzyme B (GrB).

In some aspects, the serine protease is a truncated serine proteasehaving an IIGG, IVGG or ILGG at its N-terminus. In certain aspects, theGrB polypeptide comprises an amino acid substitution or deletion at oneor more positions selected from the group consisting of Asp 37, Asn 51,Asn 84, Arg 96, Arg 100, Arg 102, Asp 150, Arg 201, Cys 210, Lys 221,Lys 222, Lys 225, or Arg 226.

In some aspects, the cytotoxic agent is a chemotherapeutic or a toxin,such as auristatin, particularly monomethylaurostatin E (MMAE). Inspecific aspects, the MMAE comprises a protease-cleavable linker, suchas citrulline-valine.

In some aspects, the sortase recognition sequence is a C-terminalsortase donor sequence or an N-terminal sortase acceptor sequence. Incertain aspects, the C-terminal sortase donor sequence is LPXT(G)_(n),such as LPETGG. In some aspects, the N-terminal sortase acceptorsequence is a poly-glycine sequence, such as GGG.

In additional aspects, the compound further comprises at least onespacer positioned between the serine protease and sortase recognitionsequence. In particular aspects, the compound comprises two spacers. Incertain aspects, the spacer comprises the G₄S (GGGGS; SEQ ID NO: 36)sequence. In other aspects, the compound does not comprise a spacer.

In certain aspects, the cytotoxic agent is granzyme B and thepolypeptide comprises a coding sequence at least 90% identical to SEQ IDNO: 2. In some aspects, the polypeptide comprises a coding sequence atleast 95%, 98%, or 99% identical to SEQ ID NO: 2.

In some aspects, the compound is conjugated to or fused with acell-targeting moiety. In certain aspects, the cell-targeting moietycomprises sortase recognition sequence, such as a C-terminal sortasedonor sequence or an N-terminal sortase acceptor sequence. In someaspects, the C-terminal sortase donor sequence is LPXT(G)_(n), such asLPETGG. In certain aspects, the N-terminal sortase recognition sequencecomprises 1 to 10 glycine residues. For example, the N-terminal sortaserecognition sequence is GGG. In some aspects, the compound is fused witha cell-targeting moiety positioned C-terminally relative to thecytotoxic agent, such as serine protease. In other aspects, the compoundis fused with a cell-targeting moiety positioned N-terminally relativeto the cytotoxic agent, such as small molecule cytotoxic agents orcertain types of serine proteases.

In some aspects, the cell-targeting moiety is a peptide or polypeptide.In certain aspects, the cell-targeting moiety is a polynucleotide. Forexample, the polynucleotide is RNA or DNA.

In certain aspects, the cell-targeting moiety is an antibody. In someaspects, the antibody is a monoclonal, chimeric antibody, Fab′, Fab,F(ab′)2, single domain antibody, Fv, single chain Fv (scFv) antibody orVHH nanobody. In certain aspects, the antibody is a human antibody, ahumanized antibody or a deimmunized antibody. For example, the antibodyis a 15A8, ZME-018, ScFvMEL, cetuximab or trastuzumab antibody.

In some aspects, the cell-targeting moiety binds to a protein,carbohydrate or lipid expressed on cancer cells. For example, thecell-targeting moiety binds to FN14 receptor, VEGFR, GP240, 5T4, HER1,HER2, CD-33, CD-38, flt1, Flk-1, CEA, FGFR3, IGFBP2 or IGF-1R. In someaspects, the cell-targeting moiety is Yoked chorionic gonadotropin(YCG).

In some aspects, the cytotoxic agent or cell-targeting moiety is furtherconjugated to an imaging agent. For example, the imaging agent can be aradionuclide, a MRI contrast agent or an ultrasound contrast agent.Thus, in some aspects, a method is provided for imaging target cells ina subject comprising administering a cell-targeting compound conjugatedto an imaging agent to the subject and imaging the target cells in thesubject.

In a further embodiment, there is provided a recombinant fusionpolypeptide comprising: (a) a recombinant cytotoxic polypeptide; (b) asortase linker and (c) a cell-targeting polypeptide, wherein the sortaselinker is positioned between the cytotoxic polypeptide and thecell-targeting polypeptide. As used herein the term “sortase linker”refers to the residual sequence after the linkage of two polypeptides bya sortase reaction the LPXT from the C-terminal sortase donor sequenceand the G or poly-G (G_(n)) from the N-terminal sortase recognitionsequence. Thus, a sortase linker can comprise the sequence LPXT(G)_(n).In certain aspects, the recombinant cytotoxic polypeptide is arecombinant serine protease. In some aspects, the recombinant serineprotease is a truncated serine protease having an IIGG, IVGG or ILGG atits N-terminus.

In some aspects, the serine protease polypeptide is a GrB polypeptide.In other aspects, the GrB polypeptide comprises an amino acidsubstitution or deletion at one or more positions selected from thegroup consisting of Asp 37, Asn 51, Asn 84, Arg 96, Arg 100, Arg 102,Asp 150, Arg 201, Cys 210, Lys 221, Lys 222, Lys 225, or Arg 226. Insome aspects, the amino acid substitution is for a residue having apolar or positively charged side chain. In certain aspects, thepolypeptide further comprises an amino acid sequence comprising a Cys,wherein the amino acid sequence is positioned C-terminally relative tothe GrB coding sequence.

In some aspects, the sortase linker has the sequence motif LPXT(G)_(n)or other sequence motif recognized by a sortase. For example, thesortase linker comprises the sequence LPETGGG.

In certain aspects, the polypeptide comprises from N-terminus toC-terminus a recombinant GrB polypeptide; a sortase linker; and a Yokedchorionic gonadotropin (YCG) polypeptide in which the beta and alphachains are fused together to form a single polypeptide. In other aspectsthe polypeptide comprises a GrB linked by sortase to a Yoked chorionicgonadotropin in which the alpha chain is N-terminal to the beta chain.In other aspects, the polypeptide is linked by sortase to a polypeptideconsisting of the beta chain of any gonadotropin and the alpha chain ofhuman chorionic gonadotropin.

In another embodiment, there is provided a method of producing atargeted compound comprising: (a) obtaining a compound comprising acytotoxic agent as provided herein and a cell-targeting moietycomprising a sortase recognition sequence; and (b) contacting thecompound and cell-targeting moiety with a transpeptidase, therebyproducing the targeted compound. In some aspects, the transpeptidase isSortase A. In certain aspects, the compound, cell-targeting moiety, andtranspeptidase are present at a ratio of about 1:1:3 to about 1:1:6,such as about 1:1:5.

In some aspects, the compound comprises an N-terminal sortase acceptorsequence and the cell-targeting moiety comprises a C-terminal sortasedonor sequence. In other aspects, the compound comprises an C-terminalsortase donor sequence and the cell-targeting moiety comprises anN-terminal sortase acceptor sequence. In certain aspects, the C-terminalsortase donor sequence is LPXT(G)_(n), such as LPETGG. In certainaspects, the N-terminal sortase acceptor sequence comprises 1 to 10N-terminal glycine residues. For example, the N-terminal sortaseacceptor sequence comprises 3 N-terminal glycine residues.

In some aspects, the cell-targeting moiety is a peptide or polypeptide.In certain aspects, the cell-targeting moiety is a polynucleotide. Forexample, the polynucleotide is RNA or DNA. In some aspects, the RNA issiRNA, miRNA or shRNA. In other aspects, the cell-targeting moietycomprises a short peptide with an N-terminal triglycine sequenceconjugated to a polynucleotide.

A further embodiment provides a composition comprising the compoundprovided herein comprising: (a) a cytotoxic agent, such as a serineprotease polypeptide; (b) a sortase linker; and (c) a cell-targetingmoiety, such as a polypeptide, in a pharmaceutically acceptable carrier.

In an even further embodiment, there is provided a polynucleotidemolecule comprising a nucleic acid sequence encoding the recombinantpolypeptide provided herein comprising a serine protease and a sortaserecognition sequence or the polypeptide provided herein comprising: (a)a recombinant serine protease polypeptide; (b) a sortase linker; and (c)a cell-targeting polypeptide. Another embodiment provides a host cellcomprising said polynucleotide sequence. In some aspects, the host cellis a mammalian cell, a yeast cell, a bacterial cell, a ciliate cell oran insect cell.

A further embodiment provides a method of manufacturing a polypeptidecomprising: (a) expressing a polynucleotide provided herein comprising arecombinant serine protease polypeptide, a sortase linker and acell-targeting polypeptide in a cell under conditions to produce theencoded polypeptide; and (b) purifying the polypeptide from the cell.

In another embodiment, there is provided a method of treating a subjectwith a cell proliferative disease comprising administering to thesubject an effective amount of a compound provided herein. In someaspects, the cell proliferative disease is an autoimmune disease. Forexample, compound may be used in the treatment of rheumatoid arthritis,psoriasis, osteoarthritis, inflammatory bowel disease, type 1 diabetes,tissue or organ rejection or multiple sclerosis. In these aspects, celltargeting compounds may be used in combination with other treatmentregimens, such as steroids.

In other aspects, the cell proliferative disease is a cancer orprecancerous condition. For example, the cell proliferative disease is:a myeloma, leukemia or lymphoma; a cancer of the lung, breast, brain,prostate, pancreas, cervix, ovary or Fallopian tube, head and neck,esophagus, liver, skin, kidney, bone, testus, colon, or bladder; or asoft or hard tissue sarcoma. In some aspects, the method furthercomprises administering at least a second anticancer therapy to thesubject. In some aspects, the second anticancer therapy is surgicaltherapy, chemotherapy, radiation therapy, gene therapy or immunotherapy.

In other embodiments, there is provided a method of treating a bacterialor viral infection comprising administering to the subject an effectiveamount of a compound provided herein.

In some specific aspects a serine protease for use according to theembodiments comprises a sequence at least about 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to granzyme B,granzyme A, granzyme H, granzyme K, granzyme M, Cathepsin G, Chymase,Myeloblastin, Kallikrein-14, Complement factor D, PRSS3 protein,Trypsin-1, Serine protease 57 or PRSSL1 protein. In certain aspects, theserine protease is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identical to a human granzyme, such asgranzyme B (GrB).

In yet a further embodiment there is a provided a polynucleotidemolecule comprising a sequence that encodes a serine proteasepolypeptide or constructs of the embodiments. In some aspects, thepolynucleotide molecule is comprised in an expression cassette operablylinked to expression control sequences (e.g., a promoter, enhancer,intron, polyadenylation signal sequence or transcription terminatorsequence). In still further aspects, the polynucleotide molecule encodesa serine protease fusion protein such as cell-targeting construct of theembodiments.

It will be understood that in certain cases, a fusion protein maycomprise additional amino acids positioned between the truncated serineprotease and the cell targeting polypeptide. In general these sequencesare interchangeably termed “linker sequences” or “linker regions.” Oneof skill in the art will recognize that linker regions may be one ormore amino acids in length and often comprise one or more glycineresidue(s) which confer flexibility to the linker. In some specificexamples, linkers for use in the current embodiments include, withoutlimitation, the 218 (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 34), the HL (EAAAK;SEQ ID NO: 35) SSG and the G₄S (GGGGS; SEQ ID NO: 36) linkers. Suchlinker sequences can be repeated 1, 2, 3, 4, 5, 6, or more times orcombined with one or more different linkers to form an array of linkersequences. For instance, in some applications, a linker region maycomprise a protease cleavage site, such as the cleavage site recognizedby an endogenous intracellular protease. In this case when the celltargeting construct is internalized into a target cell proteolyticcleavage can separate the serine protease from a cell targeting moietyand/or other polypeptide domains. As such, cell targeting constructsaccording to this embodiment may have the advantage of enhancedintracellular activity of the targeted serine protease since potentialinterference from the cell targeting polypeptide will be reduced.

Recombinant fusion polypeptides according to the embodiments maycomprise additional amino acids attached to the serine protease, thecell targeting moiety, or both. For example, additional amino acids maybe included to aid production or purification of a cell targetingconstruct. Some specific examples of amino acid sequences that may beattached to cell targeting moiety include, but are not limited to,purification tags (e.g., a T7, MBP. GST, HA, or polyHis tag),proteolytic cleavage sites, such as a thrombin or furin cleavage site,intracellular localization signals or secretion signals.

In still further aspects, a polypeptide of the embodiments furthercomprises a cell-penetrating peptide (CPP). As used herein the terms CPPand membrane translocation peptide (MTP) as used interchangeably torefer to peptide sequences that enhance the ability of a protein to beinternalized by a cell. Examples for CPPs for use according to theembodiments include, without limitation, peptide segments derived fromHIV Tat, herpes virus VP22, the Drosophila Antennapedia homeobox geneproduct, protegrin I, as well as the T1, T2, INF7 and 26 peptidesexemplified herein. In certain aspects, a cell-targeting construct ofthe embodiments comprises CPP positioned between the serine protease andthe cell-targeting moiety or positioned C-terminally relative to thecell-targeting moiety. In certain aspects a CPP is separated from aserine protease and/or a cell-targeting moiety by a linker sequence.

A cell targeting construct (e.g., comprising a cell-targeting moiety anda serine protease linked by a sortase) according to the embodiments willdesirably have two properties: (1) binding affinity for a specificpopulation of cells; and, (2) the ability to be internalized into cells.It is envisioned, however, that even cell targeting constructs that arepoorly internalized may be used in methods according to the embodiments.Methods well known to those in the art may be used to determine whethera particular cell targeting construct is internalized by target cells,for example by immunohistochemical staining or immunoblot ofintracellular extracts. It is also envisioned that, in certain cases,cell targeting moieties that cannot, by themselves, be internalized, maybe internalized in the context of the cell targeting constructsaccording to the embodiments. Cell targeting moieties for use in theembodiments include but are not limited to antibodies, growth factors,hormones, peptides, aptamers, avimers (see for example U.S. PatentPublns. 20060234299 and 20060223114, incorporated herein by reference)and cytokines. As discussed above, cell targeting moieties may beconjugated to a serine protease via a covalent or non-covalent linkage,and in certain cases the targeting construct may be a fusion protein.

In certain preferred aspects, cell targeting moieties for use in theembodiments are antibodies or fragments thereof. In general the termantibody includes, but is not limited to, polyclonal antibodies,monoclonal antibodies, single chain antibodies, humanized antibodies, adeimmunized antibodies, minibodies, dibodies, tribodies as well asantibody fragments, such as Fab′, Fab, F(ab′)2, single domain antibody,Fv, or single chain Fv (scFv) antibody single domain antibodies, VHHantibodies and antibody mimetics, such as anticalins, and any mixturethereof. In some cases the cell targeting moiety is a single chainantibody (scFv). In a related aspect, the cell targeting domain may bean avimer polypeptide. Therefore, in certain cases, the cell targetingconstructs of the embodiments are fusion proteins comprising a GrBpolypeptide and a scFv or an avimer. For example, in some very specificaspects, the GrB polypeptide is conjugated or fused to a 15A8, scFvMEL,ZME-018, scFv23, cetuximab or trastuzumab antibody. Likewise, a GrBpolypeptide may be fused or conjugated to and anti-CD-33 or anti-CD-38antibody.

Thus, in some embodiments, the invention provides a cell targetingmoiety comprising a human antibody heavy chain and light chain, whereinthe antibody light chain, heavy chain or both comprise a truncatedserine protease of the embodiments positioned C-terminally relative tothe antibody light chain and/or heavy chain. For example, the antibodycan be a human IgG, such as an IgG1.

In yet further aspects, a cell targeting moiety may be a hormone. Someexamples of hormones for use in the embodiments include, but are notlimited to, human chorionic gonadotropin, gonadotropin releasinghormone, an androgen, an estrogen, thyroid-stimulating hormone,follicle-stimulating hormone, luteinizing hormone, prolactin, growthhormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin,thyrotropin-releasing hormone, growth hormone releasing hormone,corticotropin-releasing hormone, somatostatin, dopamine, melatonin,thyroxine, calcitonin, parathyroid hormone, glucocorticoids,mineralocorticoids, adrenaline, noradrenaline, progesterone, insulin,glucagon, amylin, erythropoitin, calcitriol, calciferol,atrial-natriuretic peptide, gastrin, secretin, cholecystokinin,neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor-1, leptin,thrombopoietin or angiotensinogen. As discussed above targetingconstructs that comprise a hormone can be used in methods of targetingcell populations that comprise extracellular receptors for the indicatedhormone.

In yet still further aspects of the embodiments, cell targeting moietiesmay be cytokines. For example, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8,IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, granulocyte-colonystimulating factor, macrophage-colony stimulating factor,granulocyte-macrophage colony stimulating factor, leukemia inhibitoryfactor, erythropoietin, granulocyte macrophage colony stimulatingfactor, oncostatin M, leukemia inhibitory factor, IFN-γ, IFN-α, IFN-β,LT-β, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, TGF-β,IL 1α, IL-113, IL-1RA, MIF, TNF-like weak inducer of apoptosis (TWEAK)and IGIF may all be used as targeting moieties according to theembodiments.

From the foregoing description it will be clear to one of skill in theart that cell targeting constructs according to the embodiments maytarget particular populations of cells depending on the cell targetingmoiety that is employed. For instance, the cell targeting moiety may bean infected cell targeting moiety. In this case, the cell targetingmoiety may bind to a cellular protein that is primarily expressed on thesurface of cells that are infected by a pathogen, such as bacteria, aprotozoan or a virus. In certain other aspects, the cell targetingmoiety may bind to a factor encoded by the pathogen, such as abacterial, protozoal or viral protein. In this aspect, it is envisionedthat cell targeting constructs may be indirectly targeted to cells bybinding to a pathogen before or as it enters a target cell. Thus, thetransit of a pathogen into a cell may, in some instances, mediateinternalization of the targeting construct. In additional aspects, celltargeting moieties may bind to polypeptides encoded by the pathogen thatare expressed on the surface of infected cells. For example, in the caseof a cell infected with human immunodeficiency virus (HIV), a celltargeting moiety may bind to, for example, gp120. It is envisioned thatany of the foregoing methods may be used to limit the spread ofinfection. For example, delivery of a serine protease (e.g., GrB) to theinfected cell may induce apoptosis or sensitize a cell to undergoapoptosis.

In some aspects of the embodiments a cell-targeting moiety can bedefined as an immune cell targeting moiety. In this case, the celltargeting moiety may bind to and/or be internalized by a cell surfacemolecule that is expressed on a specific populations of immune cells.Thus, targeting a serine protease to certain types of immune cells maybe used, for example, to treat autoimmune diseases, cancers, leukemias,myelomas or lymphomas.

In still further aspects of the embodiments a cell targeting moiety canbe a cancer cell targeting moiety. It is well known that certain typesof cancer cells aberrantly express surface molecules that are unique ascompared to surrounding tissue. Thus, cell targeting moieties that bindto these surface molecules enable the targeted delivery of serineproteases specifically to the cancers cells. For example, acell-targeting moiety may bind to and be internalized by a lung, breast,brain, prostate, spleen, pancreatic, cervical, ovarian, head and neck,esophageal, liver, skin, kidney, leukemia, bone, testicular, colon,sarcoma or bladder cancer cell. Thus, the effectiveness of a cancercell-targeted serine protease may, in some cases, be contingent upon theexpression or expression level of a particular cancer marker on thecancer cell. In certain aspects, there is provided a method for treatinga cancer patient with targeted serine protease comprising identifyingwhether (or to what extent) cancer cells of the patient expresses aparticular cell surface marker and administering a targeted serineprotease therapy (optionally, in conjunction with a further anticancertherapy) to a patient identified to have a cancer expressing theparticular cell surface marker. In further aspects, the dose of atargeted serine protease therapy can be adjusted depending on theexpression level of a cell surface marker on the cancer cells.

Accordingly, in certain embodiments, there is provided a method fortreating a cell proliferative disease comprising administering acell-targeting construct according to the embodiments. As used hereinthe phrase “cell proliferative condition” includes but is not limited toautoimmune diseases, diseases in which there is uncontrolledproliferation of epithelial or mesenchymal components, cancers andprecancerous conditions. For example, methods of the embodiments may beused for the treatment of cancers such as lung, breast, brain, prostate,spleen, pancreatic, cervical, ovarian, head and neck, esophageal, liver,skin, kidney, leukemia, bone, testicular, colon, or bladder cancers. Forexample, there is provided a method for treating a skin cancer, such asa melanoma, by administration of a serine protease targeted to skincancer cells. Likewise, there is provided a method for treating a gp240positive skin cancer comprising administering a serine protease of theembodiments that comprises a scFvMEL targeting moiety.

In a further embodiment, there is provided a composition comprising acell-targeting moiety and a therapeutic agent, wherein thecell-targeting moiety comprises an R-spondin or fragment thereof. Insome aspects, the R-spondin is RSPO1 or RSPO2. In particular aspects,the R-spondin is recombinant. In certain aspects, the compositionfurther comprises an imaging agent. The composition may formulated in aliposome or nanoparticle wherein the cell-targeting moiety is conjugatedon the surface and the therapeutic agent and/or imaging agent areencapsulated within the particle.

In certain aspects, the therapeutic agent (e.g., an anti-cancer agent)is a cytotoxic agent, an antibody, a growth factor, a hormone, apeptide, an aptamer, or a cytokine. In some aspects, the cytotoxic agentis further defined as a cytotoxic polypeptide, such as a serineprotease, a Bcl-2 family member, cytochrome C, or a caspase. Inparticular aspects, the serine protease is granzyme B, granzyme A,granzyme H, granzyme K, granzyme M, Cathepsin G, Chymase, Myeloblastin,Kallikrein-14, Complement factor D, PRSS3 protein, Trypsin-1, Serineprotease 57 or PRSSL1 protein. In specific aspects, the serine proteaseis Granzyme B (GrB). In some aspects, the serine protease is a truncatedserine protease having an IIGG, IVGG or ILGG at its N-terminus. Inparticular aspects, the GrB polypeptide comprises an amino acidsubstitution or deletion at one or more positions selected from thegroup consisting of Asp 37, Asn 51, Asn 84, Arg 96, Arg 100, Arg 102,Asp 150, Arg 201, Cys 210, Lys 221, Lys 222, Lys 225, or Arg 226. Alsoprovided herein are constructs that express R-spondin and a therapeuticpolypeptide, optionally fused with a linker.

In some aspects, the cytotoxic agent is a chemotherapeutic or a toxin.In specific aspects, the toxin is auristatin, such asmonomethylaurostatin E (MMAE). In one particular aspects, MMAE comprisesa protease-cleavable linker (e.g., citrulline-valine).

In certain aspects, the antibody is further defined as a full-lengthantibody, chimeric antibody, Fab′, Fab, F(ab′)2, single domain antibody(DAB), Fv, single chain Fv (scFv), minibody, diabody, triabody, or amixture thereof.

In some aspects, the cell-targeting moiety and the therapeutic agent arechemically conjugated. In certain aspects, the cell-targeting moiety andthe therapeutic agent are comprised in a fusion polypeptide. Inparticular aspects, the cell-targeting moiety and the therapeutic agentare connected by a linker.

In yet another embodiment, there is provided a method of treating cancerin a subject comprising administering to the subject an effective amountof a composition comprising a cell-targeting moiety fused to ananti-cancer agent, wherein the cell-targeting moiety comprises anR-spondin or fragment thereof. In particular aspects, the R-spondin isRSPO1 or RSPO2. In certain aspects, the cancer is a cancer of the lungs,breast, colon, ovary, or endometrium.

In some aspects, the subject has cancer cells that express an LGRreceptor. In particular aspects, the cancer cells (e.g., cancer stemcells) overexpress an LGR receptor as compared to normal cells; thus,the anti-cancer agent is selectively delivered to cancer cells. Forexample, the LGR receptor can be LGR5 or LGR6. In particular aspects,the cancer cells (e.g., cancer stem cells) express LGR6.

In additional aspects, the method further comprises administering atleast one additional cancer therapy. In some aspects, the additionalcancer therapy is chemotherapy, surgery, radiation, gene therapy,hormone therapy, immunotherapy (e.g., immune checkpoint inhibitor), or acombination thereof. In some aspects, the additional cancer therapy andthe composition are administered concomitantly, administered insuccession, the composition is administered prior to the additionalcancer therapy, or the composition is administered subsequent to theadditional cancer therapy.

Embodiments discussed in the context of a methods and/or composition ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Schematic of the sortase reaction to form new peptide bondsbetween two proteins one of which contains LPXTGG at C-terminal end andthe other of which contains a GGG sequence at its N-terminal end.

FIG. 2: Schematic of the GrB-(G₄S)₂-LPETGG construct for use in sortasereaction.

FIG. 3: Expression of GrB-(G₄S)₂-LPETGG in individual single clones wasdetermined by Western blot analysis using anti-GrB antibody at 1:1000dilution.

FIG. 4: Structure of acceptor peptides capable of reporting on kineticsof the sortase reaction.

FIGS. 5A-5B: GrB-(G₄S)₂-LPETGG cDNA (A) (SEQ ID NO: 1) and amino acidsequence (B) (SEQ ID NO: 2).

FIGS. 6A-6C: Graphic alignments of various mammalian granzymepolypeptides and serine proteases having high homology to granzymes. Ineach case the polypeptide sequences provided are for the mature activepolypeptide (i.e., lacking the N-terminal leader sequence). (A) Figureshows an alignment of sequences for GrB from Homo sapiens (SEQ ID NO: 3;100%); Pan troglodytes (SEQ ID NO: 4; 98%); Pan paniscus (SEQ ID NO: 5;98%); Pongo abelii (SEQ ID NO: 6; 93%); Macaca nemestrina (SEQ ID NO: 7;87%); Macaca mulatta (SEQ ID NO: 8; 87%); Macaca fascicularis (SEQ IDNO: 9; 86%); Sus scrofa (SEQ ID NO: 10; 72%); Bos taurus (SEQ ID NO: 11;72%); Rattus norvegicus (SEQ ID NO: 12; 70%); and Mus musculus (SEQ IDNO: 13; 71%). Percent values in parenthesis indicate the percentidentity to mature H. sapiens GrB. The amino acid positionscorresponding to human GrB Asp 37, Asn 51, Asn84, Asp150, and Cys210 areeach indicated in bold and shaded. * next to H. sapiens indicates thatcertain sequence reads for GrB indicate a “Q” at position 35 rather thanthe “R” depicted, see e.g., NCBI accession nos. AAA75490.1 versusEAW66003.1. (B) Figure shows an alignment of sequences for variousmature Granzyme polypeptides from Homo sapiens. Sequences are shown forgranzyme B “Gzm B” (SEQ ID NO: 3), granzyme A “Gzm A” (SEQ ID NO: 14),granzyme H “Gzm H” (SEQ ID NO: 15), granzyme K “Gzm K” (SEQ ID NO: 16)and granzyme M “Gzm M” (SEQ ID NO: 17). (C) Figure shows an alignment ofsequences for serine protease polypeptides from Homo sapiens with highhomology to granzyme polypeptides. Sequences are shown for maturegranzyme B (SEQ ID NO: 3), Cathepsin G (SEQ ID NO: 18, NCBI accessionno. P08311), Chymase (SEQ ID NO: 19, NCBI accession no. P23946),Myeloblastin (SEQ ID NO: 20, NCBI accession no. P24158), Kallikrein-14(SEQ ID NO: 21, NCBI accession no. Q9P0G3), Complement factor D (SEQ IDNO: 22, NCBI accession no. K7ERG9), PRSS3 protein (SEQ ID NO: 23, NCBIaccession no. A1A508), Trypsin-1 (SEQ ID NO: 24, NCBI accession no.P07477), Serine protease 57 (SEQ ID NO: 25, NCBI accession no. Q6UWY2)and PRSSL1 protein (SEQ ID NO: 26, NCBI accession no. B7ZMF6). In thealignments “*” indicated identical amino acid positions, “:” and “.”indicate highly similar or similar amino acid positions respectively.

FIG. 7: Schematic of sortase-mediated reaction of YCG-(G₄S)₂-LPETGG withGGG-vc-MMAE.

FIG. 8: Expression of YCG-(G₄S)₂-LPETGG in individual single clones wasdetermined by Western blot analysis using anti-hCG Ab at 1:2000dilution.

FIG. 9: Western blot analysis showing successful conjugation ofYCG-LPETGG with G5K-biotin using the sortase reaction. Product increasedwith increasing concentrations of G5K-biotin.

FIG. 10: Sortase-mediated ligation of YCG-(G₄S)₂-LPETGG with GGG-vc-MMAEand subsequent reverse IMAC purification. The components of reactionmixture were analyzed by Western blot analysis using anti-MMAE andanti-hCG antibody. Lane 1: SrtA; Lane 2: YCG-LPETGG; Lane 3:GGG-vc-MMAE; Lane 4: SrtA+YCG-LPETGG+GGG-vc-MMAE; Lane 5: Flow throughof sortase reaction mixture; Lane 6: Wash; Lane 7: Elution; Lane 8:Dialyzed flow through+Wash.

FIGS. 11A-C: Expression and purification ofMBP-FLAG-RSPO1/2-(G₄S)₂-LPETGG in E. coli. (A) Schematic of theMBP-FLAG-RSPO1/2-(G₄S)₂-LPETGG construct for use in sortase reaction.(B) IMAC purification of MBP-FLAG-RSPO1-(G₄S)₂-LPETGG (see arrowdenoting purified MBP-FLAG-RSPO1-(G₄S)₂-LPETGG). (C) IMAC purificationof MBP-FLAG-RSPO2-(G₄S)₂-LPETGG. Lane 1: whole cell lysate beforeinduction; Lane 2: whole cell lysate after 4h induction; Lane 3: clearlysate; Lane 4: flow through; Lane 5: wash 1; Lane 6: wash 2; Lane 7:elution 1; Lane 8: elution 2; Lane 9: elution 3.

FIG. 12: Western blot analysis of sortase-mediated reaction ofMBP-FLAG-RSPO1/2-(G₄S)₂-LPETGG with GGG-vc-MMAE using anti-MMAEantibody. Anti-MMAE antibody was able to detect a ligated product withMW close MBP-FLAG-RSPO1/2 indicating successful ligation of MMAE to thedonor, and the yield increased with amount of acceptor MMAE input.

FIG. 13: LGR6 expression in 4 populations of HEK293T cells transfectedwith pcDNA3.1/LGR6-myc-his and selected with G418

FIG. 14: MBP-RSPO1-MMAE concentration-survival curve. Cells were exposedto increasing concentrations of the drug for 10 days and the cellviability was determined by CCK-8 assay.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. The Present Invention

Granzyme B (GrB), a serine-dependent and aspartate-specific protease, isthe major effector molecule of cellular immune defense and is releasedfrom the granules of activated cytotoxic T lymphocytes or natural killercells during their attack on other cells. Once in the target cell GrB isactivated by removal of two amino acids at the N-terminus; the activatedprotein then induces apoptosis through either caspase-dependent orindependent pathways (Wowk et al., 2004). Engineering the active form ofGrB into an immunotoxin not only confines the delivery of the toxin tothe antigen-expressing tumor cells, but also eliminates the need forproteolytic activation once internalized into the target cell. GrB hasnow been used as the warhead in multiple different immunotoxins directedat the FN14 receptor (Zhou et al., 2014), HER2 (Cao et al., 2014), VEGFR(Mohamedali et al., 2009) and melanoma antigens (Liu et al., 2006) eachof which has demonstrated nanomolar potency and substantial activity inxenograft models of human cancer. GrB is of particular interest as awarhead because only a few molecules of GrB need to reach the cytoplasmto trigger apoptosis and, as evidenced by the ability of appropriatelytriggered cytotoxic T cells, there is little resistance to this enzyme.Thus far, GrB immunotoxins have been made with the GrB already fused tothe targeting moiety which poses substantial production problems. Thus,the present invention overcome problems associated with currenttechnologies by providing methods and compositions for constructingcompounds comprising cytotoxic agents, such as serine protease or MMAE,using the sortase reaction.

In some aspects, there is provided a serine protease (e.g., GrB)cassette that can be snapped onto the N-terminal end of any of a widevariety of cell-targeting proteins using the sortase reaction. Forexample, the serine protease construct has a C-terminal sortaserecognition sequence which can be cleaved by a transpeptidase, such asSortase A. In some aspects, the sortase recognition sequence has theLPXT motif. In particular aspects, the serine protease and sortaserecognition sequence are combined with at least one spacer sequence. Inother aspects, the construct does not comprise a spacer. In someaspects, the cell-targeting protein has a polyglycine sequence at itsN-terminal.

In further aspects, there are provided methods of producing acell-targeting fusion protein by combining a serine protease with aC-terminal sortase recognition sequence, a transpeptidase and acell-targeting peptide or polypeptide with an N-terminal glycine. Inthis method, the serine protease and cell-targeting moiety are coupledtogether to form a fusion protein.

In a further aspect, a recombinant fusion protein of the embodimentscomprises from N-terminus to C-terminus a serine protease polypeptide; asortase linker; and a cell-targeting moiety such as yoked humanchorionic gonadotropin (YCG). The term “sortase linker” as used hereinrefers to the amino acid segment which results after a sortase reactionfuses a donor molecule with a C-terminal sortase donor sequence (e.g.,LPXT(G)_(n)) and a acceptor molecule with a N-terminal sortase acceptorsequence (e.g., GGG) by cleaving the Thy-Gly bond of the sortase donorsequence and forms a new peptide bond with the sortase acceptorsequence. Thus, the sortase reaction results in the loss of at least 1Gly from the sortase donor sequence and may result in the loss of 2, 3,or more Gly residues. Such constructs are exemplified herein in Example2 and demonstrate highly selective toxicity to luteinizing hormonereceptor-expressing cells. Accordingly, the fusion proteins providedherein can be used to treat cancers such as ovarian, breast, endometrialand prostate carcinomas.

II. Cytotoxic and Cytostatic Agents

A. Serine Protease Polypeptides

Certain aspects of the embodiments concern compounds that comprise acyotoxic agent, such as a serine protease, and a sortase recognitionsequence. In preferred aspects, a serine protease for use according tothe embodiments is a human or substantially human polypeptide. Forexample, the truncated serine protease can be a granzyme selected fromgranzyme B, granzyme A, granzyme H, granzyme K or granzyme M, or apolypeptide at least about 80%, 85%, 90% or 95% identical to one thesegranzyme polypeptides. In still further aspects, the serine protease isa protease from Homo sapiens having an N-terminal amino acid sequence ofIIGG, IVGG or ILGG (when in its mature, active form). For example, theserine protease can be Cathepsin G (NCBI accession no. P08311), Chymase(NCBI accession no. P23946), Myeloblastin (NCBI accession no. P24158),Kallikrein-14 (NCBI accession no. Q9P0G3), Complement factor D (NCBIaccession no. K7ERG9), PRSS3 protein (NCBI accession no. A1A508),Trypsin-1 (NCBI accession no. P07477), Serine protease 57 (NCBIaccession no. Q6UWY2) or PRSSL1 protein (NCBI accession no. B7ZMF6) or apolypeptide at least about 80%, 85%, 90% or 95% identical to one theseprotease polypeptides.

In certain cases, serine protease polypeptides or portions thereof maybe from a non-human source or may be from a homologous humanpolypeptide. For example, in the case of GrB, a polypeptide may compriseone or more amino acid substitutions to an amino acid at a correspondingposition in a Pan troglodytes; Pan paniscus; Pongo abelii; Macacanemestrina; Macaca mulatta; Macaca fascicularis; Sus scrofa; Bos taurus;Rattus norvegicus; or Mus musculus GrB (see, FIG. 6A). Likewise, agranzyme polypeptide of the embodiments may comprise one or more aminoacid substitutions to an amino acid at a corresponding position in adifferent granzyme coding sequence (see, e.g., FIG. 6B). In yet furtheraspects, a serine protease of the embodiments may comprise one or moreamino acid substitutions to an amino acid at a corresponding position ina different, homologous, serine protease coding sequence (see, e.g.,FIG. 6C). Because of the high homology shared between thesepolypeptides, such substitutions for corresponding amino acid positionsdiscussed above would be expected to result in a coding sequences that,when expressed, maintains protease activity.

In certain aspects, a serine protease for use according to theembodiments is a GrB polypeptide. Thus, one or more of the molecules foruse in the current embodiments include, but are not limited to, humanGrB polypeptide that is at least 70%, 80%, 90%, 95%, 98% or moreidentical to human GrB (SEQ ID NO: 3). In certain aspects a recombinantGrB sequence is provided wherein one or more amino acid has beensubstituted for an amino acid at a corresponding position of GrB fromanother species (other than human).

In some aspects, the serine protease is one described in U.S. Pat. No.9,096,840 or U.S. Patent Application Nos. 2014/0140976 and 2015/0010556,each incorporated herein by reference in their entirety. For example, incertain specific aspects, a granzyme for use according to theembodiments is a GrB coding sequence comprising one or more amino aciddeletions and/or substitutions relative to a human GrB sequence such asSEQ ID NO: 3 (see also NCBI accession numbers nos. AAA75490.1 andEAW66003.1, incorporated herein by reference). For example, therecombinant GrB can be at least 80% identical to SEQ ID NO: 3 (e.g., atleast about or about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to SEQ ID NO: 3). In certain aspects, a GrB polypeptidecomprises one or more amino acid substitution to a corresponding aminoacid from a GrB of a different species. For instance, a substantiallyhuman GrB polypeptide can comprise 1, 2, 3, 4, 5, or more substitutionsat amino acid positions for a corresponding amino acid from one of theGrB polypeptides provided in FIG. 6 (e.g., a primate, porcine, bovine ormurine GrB). In some aspects, the recombinant GrB comprises one or moreof the following features: (a) an amino acid substitution or deletion atthe position corresponding to Asp 37; (b) an amino acid substitution ordeletion at the position corresponding to Asp 150; (c) an amino acidsubstitution or deletion at the position corresponding to Asn 51; (d) anamino acid substitution or deletion at the position corresponding to Asn84; and/or (e) an amino acid substitution or deletion at the positioncorresponding to Cys 210. In further aspects, a GrB polypeptidecomprises two, three, four or five of the features (a)-(e). In certainaspects, a recombinant GrB is defined as a substantially un-glycosylatedGrB polypeptide.

In a further embodiment a recombinant GrB polypeptide of the embodimentscomprises one or more of the following features: (a) an amino acidsubstitution or deletion at the position corresponding to Asp 37; (b) anamino acid substitution or deletion at the position corresponding to Asn51; (c) an amino acid substitution or deletion at the positioncorresponding to Asn 84; (d) an amino acid substitution or deletion atthe position corresponding to Arg 96; (e) an amino acid substitution ordeletion at the position corresponding to Arg 100; (f) an amino acidsubstitution or deletion at the position corresponding to Arg 102; (g)an amino acid substitution or deletion at the position corresponding toAsp 150; (h) an amino acid substitution or deletion at the positioncorresponding to Arg 201; (i) an amino acid substitution or deletion atthe position corresponding to Cys 210; (j) an amino acid substitution ordeletion at the position corresponding to Lys 221; (k) an amino acidsubstitution or deletion at the position corresponding to Lys 222; (1)an amino acid substitution or deletion at the position corresponding toLys 225; and/or (m) an amino acid substitution or deletion at theposition corresponding to Arg 226. Thus, in some aspects, a recombinantpolypeptide of the embodiments comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or all 13 of the features (a)-(m).

In certain aspects, a recombinant GrB polypeptide lacks glycosylation atan amino acid position corresponding to human amino acid position Asn 51and/or Asn 84. In some aspects, a GrB polypeptide of the embodimentscomprises an amino acid substitution or deletion at a positioncorresponding to human amino acid position Asn 51 and/or Asn 84. Infurther aspects, a GrB polypeptide comprises a Arg, His, Lys, Asp, Glu,Ser, Thr, Gln, Cys, Gly, Pro, Ala, Val, Ile, Leu, Met, Phe, Tyr or Trpsubstitution at human amino acid position Asn 51 and/or Asn 84. Forexample, in one aspect, a recombinant GrB comprises an Ala, Ser, Thr,Lys or Gln substitution at a position corresponding to human amino acidposition Asn 51. Alternatively or additionally, a recombinant GrBcomprises an Ala, Ser, Thr, Arg or Gln substitution at a positioncorresponding to human amino acid position Asn 84.

In some aspects, a recombinant GrB polypeptide comprises an amino acidsubstitution or deletion at the positions corresponding to Lys 27 and/orArg 28. For example, a recombinant GrB may comprise a substitution atboth the positions corresponding to Lys 27 and Arg 28. In some cases,the substitution is selected from K27E or K27L and R28A. In stillfurther aspects, a recombinant GrB coding sequence one, two or threeamino acid substitutions or deletions at the positions corresponding to⁸²PKN⁸⁴. For example, in some specific aspects, a GrB coding sequencecomprises the sequence PVPN substituted at the positions correspondingto ⁸²PKN⁸⁴.

In further aspects, a recombinant GrB polypeptide of the embodimentscomprises an amino acid deletion or substitution (e.g., a substitutionof an amino acid having a polar side chain) at an amino acid positioncorresponding to human amino acid position Asp 37 and/or Asp 150. Thus,in some aspects a recombinant GrB polypeptide comprises a Arg, His, Lys,Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Val, Ile, Leu, Met, Phe,Tyr or Trp substitution at to human amino acid position Asp 37 and/orAsp 150. For example, a recombinant GrB can comprise a Ser, Thr, Gln,Glu or Asn substitution at a position corresponding to human amino acidposition Asp 37. Alternatively or additionally, a recombinant GrBcomprises a Ser, Thr, Gln, Glu or Asn substitution at a positioncorresponding to human amino acid position Asp 150.

In some aspects, a recombinant GrB polypeptide of the embodimentscomprises an amino acid substitution or deletion at a positioncorresponding to human amino acid position Arg 96, Arg 100, Arg 102, Arg201, and/or Arg 226. In further aspects, a GrB polypeptide comprises aAsn, His, Lys, Asp, Glu, Ser, Thr, Gln, Cys, Gly, Pro, Ala, Val, Ile,Leu, Met, Phe, Tyr or Trp substitution at human amino acid position Arg96, Arg 100, Arg 102, Arg 201, and/or Arg 226. In certain aspects, arecombinant GrB comprises a substitution at a position corresponding toArg 96, Arg 100, Arg 102, Arg 201, and/or Arg 226 for an amino acidresidue having a polar or positively charged side chain. For example, arecombinant GrB can comprise an Ala, Asn, Ser, Thr, Lys, His or Glnsubstitution at a position corresponding to human amino acid positionArg 96, Arg 100, Arg 102, Arg 201, and/or Arg 226. In still furtheraspects, a recombinant polypeptide comprises a deletion or substitutionat 2, 3, 4 or 5 of said Arg positions.

In certain aspects, a recombinant GrB polypeptide of the embodimentscomprises an amino acid substitution or deletion at a positioncorresponding to human amino acid position Lys 221, Lys 222 and/or Lys225. In further aspects, a GrB polypeptide comprises a Asn, His, Arg,Asp, Glu, Ser, Thr, Gln, Cys, Gly, Pro, Ala, Val, Ile, Leu, Met, Phe,Tyr or Trp substitution at human amino acid position Lys 221, Lys 222and/or Lys 225. In certain aspects, a recombinant GrB comprises asubstitution at a position corresponding to Lys 221, Lys 222 and/or Lys225 for an amino acid residue having a polar or positively charged sidechain. For example, a recombinant GrB can comprise an Ala, Asn, Ser,Thr, Arg, His or Gln substitution at a position corresponding to humanamino acid position Lys 221, Lys 222 and/or Lys 225. In still furtheraspects, a recombinant polypeptide comprises a deletion or substitutionat 2 or 3 of said Lys positions.

In still further aspects, a recombinant GrB polypeptide of theembodiments comprises an amino acid deletion or substitution at theposition corresponding to Cys 210. In some aspects, recombinant GrBcomprises a Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala,Val, Ile, Leu, Met, Phe, Tyr or Trp amino acid substitution at theposition corresponding to Cys 210. For example, the recombinant GrBpolypeptide can comprise an Ala, Val, Ile, Leu, Met, Ser, Thr, Asn, Phe,Tyr or Gln substitution at the position corresponding to Cys 210.

In still a further embodiment, the serine protease is a truncated serineprotease such that the leader sequence, positioned N-terminally relativeto a IIGG, IVGG or ILGG sequence has been removed or replaced with aheterologous sequence. Thus, in certain embodiments the serine proteaseis a recombinant polypeptide comprising a cleavage site that issusceptible to cleavage by a selected protease fused to a truncatedserine protease having an IIGG, IVGG or ILGG at its N-terminus, suchthat, upon cleavage of the polypeptide by the selected protease, thetruncated serine protease having an N-terminal isoleucine will bereleased from the polypeptide. In some aspects, the protease cleavagesite is a caspase, furin, granzyme B or factor Xa cleavage sequence. Insome aspects, the protease cleavage site is for an intracellular orextracellular protease. For instance, the cleavage site can be a caspase1-10 cleavage site (e.g., YEVD, WEHD, DVAD, DEHD, DEVD, DMQD, LEVD,LEHD, VEID, VEHD, IETD, LETD or IEAD), a furin cleavage site (RVRR), agranzyme B cleavage site (IEPD) or a factor Xa cleavage site((I/A)(E/D)GR).

Thus, in the case of GrB, upon protease cleavage free GrB is releasedhaving an amino terminal sequence of IIGGHEAK (SEQ ID NO: 38). Incertain aspects, the protease cleavage site is a site cleaved by amammalian intracellular protease (e.g., a protease that cleaves at theC-terminus of its recognition sequence). In some aspects, therecombinant serine protease is a GrB polypeptide and comprises thesequence YVDEVDIIGGHEAK (SEQ ID NO: 39); RVRRIIGGHEAK (SEQ ID NO: 40);RVRRIIGGHEAK (SEQ ID NO: 41); (I/A)(E/D)GRIIGGHEAK (SEQ ID NO: 42);YEVDIIGGHEAK (SEQ ID NO: 43); WEHDIIGGHEAK (SEQ ID NO: 44); DVADIIGGHEAK(SEQ ID NO: 45); DEHDIIGGHEAK (SEQ ID NO: 46); DEVDIIGGHEAK (SEQ ID NO:47); DMQDIIGGHEAK (SEQ ID NO: 48); LEVDIIGGHEAK (SEQ ID NO: 49);LEHDIIGGHEAK (SEQ ID NO: 50); VEIDIIGGHEAK (SEQ ID NO: 51); VEHDIIGGHEAK(SEQ ID NO: 52); IETDIIGGHEAK (SEQ ID NO: 53); LETDIIGGHEAK (SEQ ID NO:54) or IEADIIGGHEAK (SEQ ID NO: 55).

In additional aspects, serine protease polypeptides may be furthermodified by one or more other amino substitutions while maintainingtheir enzymatic activity. For example, amino acid substitutions can bemade at one or more positions wherein the substitution is for an aminoacid having a similar hydrophilicity. The importance of the hydropathicamino acid index in conferring interactive biologic function on aprotein is generally understood in the art (Kyte and Doolittle, 1982).It is accepted that the relative hydropathic character of the amino acidcontributes to the secondary structure of the resultant protein, whichin turn defines the interaction of the protein with other molecules, forexample, enzymes, substrates, receptors, DNA, antibodies, antigens, andthe like. Thus such conservative substitution can be made in GrB andwill likely only have minor effects on their activity. As detailed inU.S. Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (0.5); histidine −0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). These values can beused as a guide and thus substitution of amino acids whosehydrophilicity values are within ±2 are preferred, those that are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred. Thus, any of the GrB polypeptides describedherein may be modified by the substitution of an amino acid, fordifferent, but homologous amino acid with a similar hydrophilicityvalue. Amino acids with hydrophilicities within +/−1.0, or +/−0.5 pointsare considered homologous. Furthermore, it is envisioned that serineprotease sequences may be modified by amino acid deletions,substitutions, additions or insertions while retaining its enzymaticactivity.

B. Additional Cytotoxic and Cytostatic Agents

Cytotoxic proteins for use in the present disclosure may further beselected from apoptotic factors or apoptosis related proteins includingAIF, Apaf e.g. Apaf-1, Apaf-2, Apaf-3, oder APO-2 (L), APO-3 (L),Apopain, Bad, Bak, Bax, Bcl-2, Bcl-xL, Bcl-xs, bik, CAD, Calpain,Caspase e.g. Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5,Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11,ced-3, ced-9, c-Jun, c-Myc, crm A, cytochrom C, CdR1, DcR1, DD, DED,DISC, DNA-PKcs, DR3, DR4, DR5, FADD/MORT-1, FAK, Fas (Fas-ligandCD95/fas (receptor)), FLICE/MACH, FLIP, fodrin, fos, G-Actin, Gas-2,gelsolin, granzyme A/B, ICAD, ICE, JNK, lamin A/B, MAP, MCL-1, Mdm-2,MEKK-1, MORT-1, NEDD, NF-_(kappa)B, NuMa, p53, PAK-2, PARP, perforin,PITSLRE, PKCdelta, pRb, presenilin, prICE, RAIDD, Ras, RIP,sphingomyelinase, thymidinkinase from herpes simplex, TRADD, TRAF2,TRAIL-R1, TRAIL-R2, TRAIL-R3, and transglutaminase.

In further embodiments, the cytotoxic agent may be selected frombispecific antibodies and bioactive compounds including nucleic acidslike DNA, mRNA, siRNA, and fragments of these; pharmaceutical compoundssuch as various therapeutic drugs; and radionuclides and cytotoxins,which can be targeted to a desired tissue or cell by the targetingmoiety. These agents may act while they remain conjugated to thetargeting protein or a portion thereof, or they may first detach fromthe targeting protein if the linking group is one that can readilycleave in vivo.

Suitable cytotoxic agents for use with the present disclosure includemicrotubule inhibitors, topoisomerase I inhibitors, intercalatingagents, inhibitors of intracellular signaling pathways, kinaseinhibitors, transcription inhibitors such as siRNAs, aRNAs, and miRNAs,and DNA minor groove binders. The cytotoxic agents may include compoundclasses such as maytansinoids, auristatins, amanitins, calicheamycins,psymberins, duocarmycins, anthracyclins, camptothecins, doxoru bicins,taxols, and pyrrolobenzodiazepines. Specific examples of cytotoxicagents include paclitaxel, docetaxel, etoposide, tenoposide,vincristine, vinblastine, colchicine, doxorubicin, daunorubicin,mithramycin, actinomycin, glucorticoids, puromycin, epirubicin,cyclophosphamide, methotrexate, cytarabine, f-fluorouracil, platins,streptozotocin, minomycin C, anthracyclines, dactinomycin oractinomycin, bleomycin, mithramycin, anthramycin, duocarmycins,ifosfamide, mitoxantrone, daunomycin, carminomycin, animoterin,melphalan, esperamicins, lexitropsins, auristatins (e.g., auristatin E,auristatin F, AEB, AEVB, AEFP, MMAE, MMAF), eleuthorobin, netropsin,podophyllotoxins, maytansiods including maytansine and DM1, andcombretestatins.

II. Sortases

Certain embodiments concern sortase recognition sequences and thecorresponding sortases. In some aspects, sortase-catalyzedtransacylation reactions allow the preparation of head-to-tailprotein-protein fusions under native conditions, with excellentspecificity and in near-quantitative yields (Popp M W, Ploegh H L (2011)Making and Breaking Peptide Bonds: Protein Engineering Using Sortase.Angew Chem Int Ed 50:5024-5032; Guimaraes C P et al. (2011)Identification of host cell factors required for intoxication throughuse of modified cholera toxin. J Cell Biol 195:751-764; and Popp M W,Antos J M, Grotenbreg G M, Spooner E, Ploegh H L (2007) Sortagging: aversatile method for protein labeling. Nat Chem Biol 3:707-708; theentire contents of each of which are incorporated herein by reference).

Sortases, sortase-mediated transacylation reactions, and their use intransacylation (sometimes also referred to as transpeptidation) forprotein engineering are well known to those of skill in the art (see,e.g., Ploegh et al., International Patent Application PCT/US2010/000274,and Ploegh et al., International Patent Application PCT/US2011/033303,the entire contents of each of which are incorporated herein byreference). In general, the transpeptidation reaction catalyzed bysortase results in the ligation of species containing a transamidaserecognition motif with those bearing one or more N-terminal glycineresidues. In some embodiments, the sortase recognition motif is an LPXTmotif or an LPXT(G)_(n) motif. As is known in the art, the substitutionof the C-terminal residue of the recognition sequence with a moietyexhibiting poor nucleophilicity once released from the sortase providesfor a more efficient ligation.

Sortase-mediated transacylation reactions are catalyzed by thetransamidase activity of sortase. A transamidase is an enzyme that canform a peptide linkage (i.e., amide linkage) between an acyl donorcompound and a nucleophilic acyl acceptor containing a NH₂—CH₂-moiety.In some embodiments, the sortase is sortase A (SrtA). However, it shouldbe noted that any sortase, or transamidase catalyzing a transacylationreaction can be used in embodiments of this invention.

In some embodiments, the sortase, or transamidase, recognition sequenceis LPXT (SEQ ID NO: 56), wherein X is a standard or non-standard aminoacid. In some embodiments, X is selected from D, E, A, N, Q, K, or R. Insome embodiments, the recognition sequence is selected from LPXT (SEQ IDNO: 56), SPXT (SEQ ID NO: 57), LAXT (SEQ ID NO: 58), LSXT (SEQ ID NO:59), NPXT (SEQ ID NO: 60), VPXT (SEQ ID NO: 61), IPXT (SEQ ID NO: 62),and YPXR (SEQ ID NO: 63). In some embodiments, X is selected to match anaturally occurring transamidase recognition sequence. Variant sortaserecognition sequences are known and described in PCT internationalpatent application WO 2013003555, U.S. Pat. No. 7,238,489 and U.S.Patent Application publication 20140030697, which are fully incorporatedby reference herein in their entirety. Examples of other sortaserecognition sequences, include, but are not limited to LPKTG (SEQ ID NO:90), LPATG (SEQ ID NO: 64), LPNTG (SEQ ID NO: 65), LPETG (SEQ ID NO:66), LPXAG (SEQ ID NO: 67), LPNAG (SEQ ID NO: 68), LPXTA (SEQ ID NO:69), LPNTA (SEQ ID NO: 70), LGXTG (SEQ ID NO: 71), LGATG (SEQ ID NO:72), IPXTG (SEQ ID NO: 73), IPNTG (SEQ ID NO: 74), and IPETG (SEQ ID NO:75). Further examples of sortase recognition sequences, include, but arenot limited to LPKTGG (SEQ ID NO: 76), LPATGG (SEQ ID NO: 77), LPNTGG(SEQ ID NO: 78), LPETGG (SEQ ID NO: 79), LPXAGG (SEQ ID NO: 80), LPNAGG(SEQ ID NO: 81), LPXTAG (SEQ ID NO: 82), LPNTAG (SEQ ID NO: 83), LGXTGG(SEQ ID NO: 84), LGATGG (SEQ ID NO: 85), IPXTGG (SEQ ID NO: 86), IPNTGG(SEQ ID NO: 87), and IPETGG (SEQ ID NO: 88).

In some embodiments, the coding sequence of sortase recognition isoperably linked to the coding sequence of the serine protease via alinker. Any suitable linker known to one of skilled in the art can beused. In a particular embodiment, the linker is a (GGS) or (G₄S) linker.The (G₄S) linker facilitates the sortase domain to have theconformational freedom to recognize the sortase recognition motif.

III. Cell Targeting Moieties

As discussed above cell targeting moieties according to the embodimentsmay be, for example, an antibody, a growth factor, a hormone, a peptide,an aptamer or a cytokine. For instance, a cell targeting moietyaccording the embodiments may bind to a skin cancer cell such as amelanoma cell. It has been demonstrated that the gp240 antigen isexpressed in a variety of melanomas but not in normal tissues. Thus, incertain aspects of the embodiments, there is provided a cell targetingconstruct comprising an GrB and a cell targeting moiety that binds togp240. In some instances, the gp240 binding molecule may be an antibody,such as the ZME-018 (225.28S) antibody or the 9.2.27 antibody. In aneven more preferred embodiment, the gp240 binding molecule may be asingle chain antibody such as the scFvMEL antibody. Therefore, in a veryspecific embodiment of the invention, there is provided a cell targetingconstruct comprising human GrB conjugated to scFvMEL.

In yet further specific embodiments of the invention, cell targetingconstructs may be directed to breast cancer cells. For example celltargeting moieties that bind to Her-2/neu, such as anti-Her-2/neuantibodies may conjugated to GrB. One example of such a cell targetingconstruct is a fusion protein comprising the single chain anti-Her-2/neuantibody scFv23 and GrB. Other scFv antibodies such as scFv(FRP5) thatbind to Her-2/neu may also be used in the compositions and methods ofthe current embodiments (von Minckwitz et al., 2005).

In certain additional embodiments, it is envisioned that cancer celltargeting moieties bind to multiple types of cancer cells. For example,the 8H9 monoclonal antibody and the single chain antibodies derivedtherefrom bind to a glycoprotein that is expressed on breast cancers,sarcomas and neuroblastomas (Onda et al., 2004). Another example are thecell targeting agents described in U.S. patent application no.2004005647 and in Winthrop et al., 2003 that bind to MUC-1, an antigenthat is expressed on a variety cancer types. Thus, it will be understoodthat in certain embodiments, cell targeting constructs according theembodiments may be targeted against a plurality of cancer or tumortypes.

Additionally, certain cell surface molecules are highly expressed intumor cells, including hormone receptors such as human chorionicgonadotropin receptor and gonadotropin releasing hormone receptor(Nechushtan et al., 1997). Therefore, the corresponding hormones may beused as the cell-specific targeting moieties in cancer therapy.

Since a large number of cell surface receptors have been identified inhematopoietic cells of various lineages, ligands or antibodies specificfor these receptors may be used as cell-specific targeting moieties. IL2may also be used as a cell-specific targeting moiety in a chimericprotein to target IL2R+ cells. Alternatively, other molecules such asB7-1, B7-2 and CD40 may be used to specifically target activated T cells(The Leucocyte Antigen Facts Book, 1993, Barclay et al. (eds.), AcademicPress). Furthermore, B cells express CD19, CD40 and IL4 receptor and maybe targeted by moieties that bind these receptors, such as CD40 ligand,IL4, IL5, IL6 and CD28. The elimination of immune cells such as T cellsand B cells is particularly useful in the treatment of autoimmunity,hypersensitivity, transplantation rejection responses and in thetreatment of lymphoid tumors. Examples of autoimmune diseases aremultiple sclerosis, rheumatoid arthritis, insulin-dependent diabetesmellitus, systemic lupus erythemotisis, scleroderma, and uviatis. Morespecifically, since myelin basic protein is known to be the major targetof immune cell attack in multiple sclerosis, this protein may be used asa cell-specific targeting moiety for the treatment of multiple sclerosis(WO 97/19179; Becker et al., 1997).

Other cytokines that may be used to target specific cell subsets includethe interleukins (IL 1 through IL15), granulocyte-colony stimulatingfactor, macrophage-colony stimulating factor, granulocyte-macrophagecolony stimulating factor, leukemia inhibitory factor, tumor necrosisfactor, transforming growth factor, epidermal growth factor,insulin-like growth factors, and/or fibroblast growth factor (Thompson(ed.), 1994, The Cytokine Handbook, Academic Press, San Diego). In someaspects, the targeting polypeptide is a cytokine that bind to the Fn14receptor, such as TWEAK (see, e.g., Winkles 2008; Zhou et al., 2011 andBurkly et al., 2007, incorporated herein by reference).

A skilled artisan recognizes that there are a variety of knowncytokines, including hematopoietins (four-helix bundles) (such as EPO(erythropoietin), IL-2 (T-cell growth factor), IL-3 (multicolony CSF),IL-4 (BCGF-1, BSF-1), IL-5 (BCGF-2), IL-6 IL-4 (IFN-β2, BSF-2, BCDF),IL-7, IL-8, IL-9, IL-11, IL-13 (P600), G-CSF, IL-15 (T-cell growthfactor), GM-CSF (granulocyte macrophage colony stimulating factor), OSM(OM, oncostatin M), and LIF (leukemia inhibitory factor)); interferons(such as IFN-γ, IFN-α, and IFN-β); immunoglobin superfamily (such asB7.1 (CD80), and B7.2 (B70, CD86)); TNF family (such as TNF-α(cachectin), TNF-β (lymphotoxin, LT, LT-α), LT-β, CD40 ligand (CD40L),Fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), and4-1BBL)); and those unassigned to a particular family (such as TGF-β, IL1α, IL-1β, IL-1 RA, IL-10 (cytokine synthesis inhibitor F), IL-12 (NKcell stimulatory factor), MIF, IL-16, IL-17 (mCTLA-8), and/or IL-18(IGIF, interferon-γ inducing factor)). Furthermore, the Fc portion ofthe heavy chain of an antibody may be used to target Fcreceptor-expressing cells such as the use of the Fc portion of an IgEantibody to target mast cells and basophils.

Furthermore, in some aspects, the cell-targeting moiety may be a peptidesequence or a cyclic peptide. Examples, cell- and tissue-targetingpeptides that may be used according to the embodiments are provided, forinstance, in U.S. Pat. Nos. 6,232,287; 6,528,481; 7,452,964; 7,671,010;7,781,565; 8,507,445; and 8,450,278, each of which is incorporatedherein by reference.

Over the past few years, several monoclonal antibodies have beenapproved for therapeutic use and have achieved significant clinical andcommercial success. Much of the clinical utility of monoclonalantibodies results from the affinity and specificity with which theybind to their targets, as well as long circulating life due to theirrelatively large size. Monoclonal antibodies, however, are not wellsuited for use in indications where a short half-life is advantageous orwhere their large size inhibits them physically from reaching the areaof potential therapeutic activity.

Thus, in highly preferred embodiments, cell targeting moieties areantibodies or avimers. Antibodies and avimers can be generated tovirtually any cell surface marker thus, providing a method for targetedto delivery of GrB to virtually any cell population of interest. Methodsfor generating antibodies that may be used as cell targeting moietiesare detailed below. Methods for generating avimers that bind to a givencell surface marker are detailed in U.S. Patent Applns. 20060234299 and20060223114, each incorporated herein by reference.

A. Antibodies and Antibody-Like Targeting Moieties

As indicated above in some aspects the cell-targeting moiety is anantibody. As used herein, the term “antibody” is intended to includeimmunoglobulins and fragments thereof which are specifically reactive tothe designated protein or peptide, or fragments thereof. Suitableantibodies include, but are not limited to, human antibodies, primatizedantibodies, de-immunized antibodies, chimeric antibodies, bi-specificantibodies, humanized antibodies, conjugated antibodies (i.e.,antibodies conjugated or fused to other proteins, radiolabels,cytotoxins), Small Modular ImmunoPharmaceuticals (“SMIPs™”), singlechain antibodies, cameloid antibodies, antibody-like molecules (e.g.,anticalins), and antibody fragments. As used herein, the term“antibodies” also includes intact monoclonal antibodies, polyclonalantibodies, single domain antibodies (e.g., shark single domainantibodies (e.g., IgNAR or fragments thereof)), multispecific antibodies(e.g., bi-specific antibodies) formed from at least two intactantibodies, and antibody fragments so long as they exhibit the desiredbiological activity. In some aspects, the antibody can be a VHH (i.e.,an antigen-specific VHH) antibody that comprises only a heavy chain. Forexample, such antibody molecules can be derived from a llama or othercamelid antibody (e.g., a camelid IgG2 or IgG3, or a CDR-displayingframe from such camelid Ig) or from a shark antibody. Antibodypolypeptides for use herein may be of any type (e.g., IgG, IgM, IgA, IgDand IgE). Generally, IgG and/or IgM are preferred because they are themost common antibodies in the physiological situation and because theyare most easily made in a laboratory setting.

As used herein, an “antibody fragment” includes a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, Fc and Fv fragments; triabodies; tetrabodies; linearantibodies; single-chain antibody molecules; and multi specificantibodies formed from antibody fragments. The term “antibody fragment”also includes any synthetic or genetically engineered protein that actslike an antibody by binding to a specific antigen to form a complex. Forexample, antibody fragments include isolated fragments, “Fv” fragments,consisting of the variable regions of the heavy and light chains,recombinant single chain polypeptide molecules in which light and heavychain variable regions are connected by a peptide linker (“ScFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region. In some aspects, theantibody fragment can be a VHH antibody.

“Mini-antibodies” or “minibodies” are also contemplated for use with thepresent embodiments. Minibodies are sFv polypeptide chains which includeoligomerization domains at their C-termini, separated from the sFv by ahinge region (Pack et al., 1992). The oligomerization domain comprisesself-associating α-helices, e.g., leucine zippers, that can be furtherstabilized by additional disulfide bonds. The oligomerization domain isdesigned to be compatible with vectorial folding across a membrane, aprocess thought to facilitate in vivo folding of the polypeptide into afunctional binding protein. Generally, minibodies are produced usingrecombinant methods well known in the art. See, e.g., Pack et al.(1992); Cumber et al. (1992).

In some cases antibody-like molecules are protein scaffolds that can beused to display antibody CDR domains. The origin of such proteinscaffolds can be, but is not limited to, the structures selected among:fibronectin (see, e.g., U.S. Patent Publn. No. 20090253899, incorporatedherein by reference) and preferentially fibronectin type III domain 10,protein Z arising from domain B of protein A of Staphylococcus aureus,thioredoxin A or proteins with a repeated motif such as the “ankyrinrepeat” (Kohl et al., 2003), the “armadillo repeat”, the “leucine-richrepeat” and the “tetratricopeptide repeat.” The techniques for preparingand using various antibody-based constructs and fragments are well knownin the art. Additional antibody-like molecules, such as anti-calins aredescribed in detail in US Patent Publication Nos. 20100285564,20060058510, 20060088908, 20050106660, PCT Publication No. WO2006/056464and (Skerra, 2001), incorporated herein by reference.

Antibody-like binding peptidomimetics are also contemplated in thepresent embodiments. Liu et al. (2003) describe “antibody like bindingpeptidomimetics” (ABiPs), which are peptides that act as pared-downantibodies and have certain advantages of longer serum half-life as wellas less cumbersome synthesis methods. Likewise, in some aspects,antibody-like molecules are cyclic or bicyclic peptides. For example,methods for isolating antigen-binding bicyclic peptides (e.g., by phagedisplay) and for using such peptides are provided in U.S. Patent Publn.20100317547, incorporated herein by reference.

Monoclonal antibodies (MAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production. Embodiments of theinvention provide monoclonal antibodies of the human, murine, monkey,rat, hamster, rabbit and chicken origin. Due to the ease of preparationand ready availability of reagents, murine monoclonal antibodies willoften be preferred.

“Humanized” antibodies are also contemplated, as are chimeric antibodiesfrom mouse, rat, or other species, bearing human constant and/orvariable region domains, bispecific antibodies, recombinant andengineered antibodies and fragments thereof. As used herein, the term“humanized” immunoglobulin refers to an immunoglobulin comprising ahuman framework region and one or more CDR's from a non-human (usually amouse or rat) immunoglobulin. The non-human immunoglobulin providing theCDR's is called the “donor” and the human immunoglobulin providing theframework is called the “acceptor”. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin. Methods for humanizing antibodies such as those providedhere are well known in the art, see, e.g., Harvey et al., 2004,incorporated herein by reference.

B. R-Spondins (RSPOs)

The LGR family of G-protein-coupled 7-transmembrane spanning receptorscontains 8 members all of which have large extracellular domainsconsisting of up to 18 copies of a leucine-rich repeat motif. The 8receptors fall into 3 groups. The first consists of LGR1 which is theFSH receptor, LGR2 the LH receptor, and LGR3 the TSH receptor. Thesecond consists of LGR4, LGR5, and LGR6 which are receptors for theR-spondins (RSPOs) and the third group contains LGR7 and LGR8 which arereceptors for relaxin and the insulin-like 3 protein, respectively. LGR5and LGR6 are the best defined markers for stem cells in the gut (LGR5)and skin and Fallopian tube epithelium (LGR6), respectively. LGR5 wasshown to be positively regulated by the Wnt signaling pathway thatcontrols the proliferation of the stem cells that form the epithelium ofthe colon, small intestine and stomach. During embryonic developmentLGR5 is expressed in multiple tissues, but in the adult its expressionis very restricted to rare cells in the gut, breast, ovary, testis, hairfollicles, brain and eye. Using a genetic marking technique, cells thatexpress LGR5 were found to function as stem cells capable of giving riseto all the other types of cells found in the epithelium of the colon andstomach. In contrast to LGR5, LGR6 is not regulated by Wnt signaling. InLGR6-LacZ^(LacZ) knock-in mice expression was found to be limited torare cells in the brain, breast, lung and hair follicles. Lineagemapping has shown that LGR6-positive cells residing in the bulb of thehair follicle are located in a different position than the LGR5-positivecells, and that they give rise to the epidermis and sebaceous glands.Subsequent studies demonstrated that LGR6-expressing cells are the stemcells that generate the new skin needed during the wound healingprocess. There is also evidence that LGR6 is uniquely expressed by tumorstem cells. LGR6 was found to mark the subpopulation of cells isolatedfrom human lung adenocarcinomas that are capable of forming new tumorsin injected into mice.

LGR5 and LGR6 are expressed in many types of tumors including cancers ofthe breast, colon and endometrium. There are several lines of evidencesuggesting that LGR6 rather than LGR5 uniquely identifies stem cells inthe Fallopian tube epithelium (FTE) and in ovarian cancers. Thus, LGR6appears to stem cells in tumors arising from the FTE. Therefore,embodiments of the present disclosure concern the use of LGR6 as atarget of tumor stem cells as it is expressed on the cell surface whereit is potentially accessible to antibodies and other kinds oftumor-targeting toxins.

R-spondins (RSPOs) are the ligands for LGR5 and LGR6. RSPO are a groupof 4 cysteine-rich secreted paralogs (R-spondin1-4). They share anoverall similarity of 40-60% sequence homology and domain architecture.All 4 RSPO family members contain an N-terminal secretory signalpeptide, 2 tandem furin-like cysteine-rich (Fu-CRD) domains, athrombospondin type1 repeat (TSP) domain, and a C-terminal basic aminoacid-rich (BR) domain. RSPO1 and RSPO2 have been identified as theligands for both LGR5 and LGR6 receptors to which they bind with highaffinity. Therefore, certain embodiments of the present disclosureconcern the use of RSPOs to target a therapeutic agent to cells whichtumor stem cells which express LGR6. In one particular aspect, RSPO1and/or RSPO2 are linked to a cytotoxic agent, such as the toxinmonomethylaurostatin E (MMAE), to selectively target tumors that expresshigh levels of the LGR6.

IV. Fusion Proteins and Conjugates

A. Linkers

A variety of linkers can be used in constructs, such as truncated serineprotease constructs, of the embodiments. In some aspects a linker can bea random string of one or more amino acids (e.g., 2, 3, 4, 5, 10, 15, 20or more amino acids). Some specific linkers for use according theembodiments include the 218 (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 34), the HL(EAAAK; SEQ ID NO: 35) and the G₄S (GGGGS; SEQ ID NO: 36) linkers (e.g.,Robinson et al., 1998; Arai et al., 2004 and Whitlow et al., 1993, eachincorporated herein by reference).

In further aspects, a linker can serve as a way of separating differentdomains of a polypeptide construct, such as by proteolytic cleavage. Forexample, a linker region may comprise a protease cleavage site, such asthe cleavage site recognized by an endogenous intracellular protease. Instill further aspects, a protease cleavage site can be a site that isonly cleaved in certain cell types (e.g., a site cleaved by a viralprotease, such as HIV protease, which is only cleaved in infectedcells). Example of protease cleavage site for use according to theembodiments include, without limitation, thrombin, furin (Goyal et al.,2000) and caspase cleavage sites.

The cell targeting constructs of the embodiments may be joined by avariety of conjugations or linkages that have been previously describedin the art. In one example, a biologically-releasable bond, such as aselectively-cleavable linker or amino acid sequence may be used. Forinstance, peptide linkers that include a cleavage site for an enzymepreferentially located or active within a tumor environment arecontemplated. For example, linkers that are cleaved by urokinase,plasmin, thrombin, Factor IXa, Factor Xa, or a metalloproteinase, suchas collagenase, gelatinase, or stromelysin. In a preferred embodiment, alinker that is cleaved by an intracellular proteinase is preferred,since this will allow the targeting construct to be internalized intactinto targeted cells prior to cleavage.

Amino acids such as selectively-cleavable linkers, synthetic linkers, orother amino acid sequences such as the glycine rich linkers aredescribed above and may be used to separate proteinaceous components. Insome specific examples linkers for use in the current embodimentsinclude the 218 linker (GSTSGSGKPGSGQGSTKG) (SEQ ID NO: 37) or the G₄Slinker (GGGGS) (SEQ ID NO: 36). Additionally, while numerous types ofdisulfide-bond containing linkers are known that can successfully beemployed to conjugate the GrB with a cell targeting moiety, certainlinkers will generally be preferred over other linkers, based ondiffering pharmacologic characteristics and capabilities. For example,linkers that contain a disulfide bond that is sterically “hindered” areto be preferred, due to their greater stability in vivo, thus preventingrelease of the toxin moiety prior to binding at the site of action.

C. Conjugates

Additionally, any other linking/coupling agents and/or mechanisms knownto those of skill in the art can be used to combine the components ofthe present embodiments, such as, for example, antibody-antigeninteraction, avidin biotin linkages, amide linkages, ester linkages,thioester linkages, ether linkages, thioether linkages, phosphoesterlinkages, phosphoramide linkages, anhydride linkages, disulfidelinkages, ionic and hydrophobic interactions, bispecific antibodies andantibody fragments, or combinations thereof.

It is contemplated that a cross-linker having reasonable stability inblood will be employed. Numerous types of disulfide-bond containinglinkers are known that can be successfully employed to conjugatetargeting and therapeutic/preventative agents. Linkers that contain adisulfide bond that is sterically hindered may prove to give greaterstability in vivo, preventing release of the targeting peptide prior toreaching the site of action. These linkers are thus one group of linkingagents.

Another cross-linking reagent is SMPT, which is a bifunctionalcross-linker containing a disulfide bond that is “sterically hindered”by an adjacent benzene ring and methyl groups. It is believed thatsteric hindrance of the disulfide bond serves a function of protectingthe bond from attack by thiolate anions such as glutathione which can bepresent in tissues and blood, and thereby help in preventing decouplingof the conjugate prior to the delivery of the attached agent to thetarget site.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amines (e.g., the epsilon amino group oflysine). Another possible type of cross-linker includes thehetero-bifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reactswith primary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Thorpe et al., 1987). The use of suchcross-linkers is well understood in the art. Another embodiment involvesthe use of flexible linkers.

U.S. Pat. No. 4,680,338, describes bifunctional linkers useful forproducing conjugates of ligands with amine-containing polymers and/orproteins, especially for forming antibody conjugates with chelators,drugs, enzymes, detectable labels and the like. U.S. Pat. Nos. 5,141,648and 5,563,250 disclose cleavable conjugates containing a labile bondthat is cleavable under a variety of mild conditions.

U.S. Pat. No. 5,856,456 provides peptide linkers for use in connectingpolypeptide constituents to make fusion proteins, e.g., single chainantibodies. The linker is up to about 50 amino acids in length, containsat least one occurrence of a charged amino acid (preferably arginine orlysine) followed by a proline, and is characterized by greater stabilityand reduced aggregation. U.S. Pat. No. 5,880,270 disclosesaminooxy-containing linkers useful in a variety of immunodiagnostic andseparative techniques.

D. Cell Penetrating and Membrane Translocation Peptides

Furthermore, in certain aspects, library sequences can include segmentsof sequence that encode polypeptides having a known function, such as acell-binding domain or cell penetrating peptide (CPP) in the ORFsequence along with sequence derived from cDNA or randomized sequence(i.e., to generate an ORF encoding a fusion protein). Thus, in certainaspects, DNA molecules of the embodiments comprise an ORF that comprisesa CPP coding sequence along with a segment of library sequence (such asrandomized sequence), 5′ of the CPP coding sequence 3′ of the CPP codingsequence or both. As used herein the terms “cell penetrating peptide”and “membrane translocation domain” are used interchangeably and referto segments of polypeptide sequence that allow a polypeptide to crossthe cell membrane (e.g., the plasma membrane in the case a eukaryoticcell). Examples of CPP segments include, but are not limited to,segments derived from HIV Tat (e.g., GRKKRRQRRRPPQ; SEQ ID NO: 27),herpes virus VP22, the Drosophila Antennapedia homeobox gene product,protegrin I, Penetratin (RQIKIWFQNRRMKWKK; SEQ ID NO: 28) or melittin(GIGAVLKVLTTGLPALISWIKRKRQQ; SEQ ID NO: 29). In certain aspects the CPPcomprises the T1 (TKIESLKEHG; SEQ ID NO: 30), T2 (TQIENLKEKG; SEQ ID NO:31), 26 (AALEALAEALEALAEALEALAEAAAA; SEQ ID NO: 32) or INF7(GLFEAIEGFIENGWEGMIEGWYGCG; SEQ ID NO: 33) CPP sequence.

V. Administration and Pharmaceutical Formulations

In some embodiments, an effective amount of a cell targeting constructis administered to a cell. In other embodiments, a therapeuticallyeffective amount of the targeting construct is administered to anindividual for the treatment of disease. The term “effective amount” asused herein is defined as the amount of the cell targeted cytotoxicagent, such as truncated serine protease, particularly GrB, of thepresent embodiments that is necessary to result in a physiologicalchange in the cell or tissue to which it is administered either whenadministered alone or in combination with a cytotoxic therapy. The term“therapeutically effective amount” as used herein is defined as theamount of the targeting molecule of the present embodiments thateliminate, decrease, delay, or minimize adverse effects of a disease,such as cancer. A skilled artisan readily recognizes that, in manycases, cell targeted cytotoxic agents may not provide a cure but mayonly provide partial benefit, such as alleviation or improvement of atleast one symptom. In some embodiments, a physiological change havingsome benefit is also considered therapeutically beneficial. Thus, insome embodiments, an amount of cell targeted cytotoxic agents, such asserine protease (e.g., GrB) that provides a physiological change isconsidered an “effective amount” or a “therapeutically effectiveamount.” It will additionally be clear that a therapeutically effectiveamount may be dependent upon the inclusion of additional therapeuticregimens tat administered concurrently or sequentially. Thus it will beunderstood that in certain embodiments a physical change may constitutean enhanced effectiveness of a second therapeutic treatment.

The cell targeting compounds of the embodiments may be administered to asubject per se or in the form of a pharmaceutical composition for thetreatment of cancer, autoimmunity, transplantation rejection,post-traumatic immune responses and infectious diseases, for example bytargeting viral antigens, such as gp120 of HIV. More specifically, thetargeted compounds may be useful in eliminating cells involved in immunecell-mediated disorder, including lymphoma; autoimmunity,transplantation rejection, graft-versus-host disease, ischemia andstroke. Pharmaceutical compositions comprising the proteins of theembodiments may be manufactured by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the proteins into preparations which canbe used pharmaceutically. Proper formulation is dependent upon the routeof administration chosen.

In preferred embodiments, cancer cells may be treated by methods andcompositions of the embodiments. Cancer cells that may be treated withcell targeting constructs according to the embodiments include but arenot limited to cells from the bladder, blood, bone, bone marrow, brain,breast, colon, esophagus, gastrointestine, gum, head, kidney, liver,lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue,or uterus. In addition, the cancer may specifically be of the followinghistological type, though it is not limited to these: neoplasm,malignant; carcinoma; carcinoma, undifferentiated; giant and spindlecell carcinoma; small cell carcinoma; papillary carcinoma; squamous cellcarcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrixcarcinoma; transitional cell carcinoma; papillary transitional cellcarcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; maligmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignantlymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular; mycosis fungoides; other specifiednon-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mastcell sarcoma; immunoproliferative small intestinal disease; leukemia;lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcomacell leukemia; myeloid leukemia; basophilic leukemia; eosinophilicleukemia; monocytic leukemia; mast cell leukemia; megakaryoblasticleukemia; myeloid sarcoma; and hairy cell leukemia.

In preferred embodiments systemic formulations of the cell targetingcompounds are contemplated. Systemic formulations include those designedfor administration by injection, e.g. subcutaneous, intravenous,intramuscular, intrathecal or intraperitoneal injection, as well asthose designed for transdermal, transmucosal, inhalation, oral orpulmonary administration. In the most preferred embodiments, the celltargeted cytotoxic agent is delivered by direct intravenous orintratumoral injection.

For injection, the proteins of the embodiments may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Alternatively, the proteins may be in powder form for constitution witha suitable vehicle, e.g., sterile pyrogen-free water, before use.

A. Effective Dosages

The cell targeted cytotoxic agent, such as serine protease, of theembodiments will generally be used in an amount effective to achieve theintended purpose. For use to treat or prevent a disease condition, themolecules of the embodiments, or pharmaceutical compositions thereof,are administered or applied in a therapeutically effective amount. Atherapeutically effective amount is an amount effective to ameliorate orprevent the symptoms, or prolong the survival of, the patient beingtreated. Determination of a therapeutically effective amount is wellwithin the capabilities of those skilled in the art, especially in lightof the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the molecules which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5to 1 mg/kg/day. Therapeutically effective serum levels may be achievedby administering multiple doses each day.

In cases of local administration or selective uptake, the effectivelocal concentration of the proteins may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

The amount of molecules administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The therapy may be repeated intermittently while symptoms detectable oreven when they are not detectable. The therapy may be provided alone orin combination with other drugs. In the case of autoimmune disorders,the drugs that may be used in combination with serine proteaseconstructs of the embodiments include, but are not limited to, steroidand non-steroid anti-inflammatory agents.

B. Toxicity

Preferably, a therapeutically effective dose of the cell targetedcytotoxic agent, such as GrB, described herein will provide therapeuticbenefit without causing substantial toxicity.

Toxicity of the molecules described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Proteinswhich exhibit high therapeutic indices are preferred. The data obtainedfrom these cell culture assays and animal studies can be used informulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (See,e.g., Fingl et al., 1975).

C. Pharmaceutical Preparations

Pharmaceutical compositions of the present embodiments comprise aneffective amount of one or more the present compounds and at least oneadditional agent dissolved or dispersed in a pharmaceutically acceptablecarrier. The phrases “pharmaceutical or pharmacologically acceptable”refers to molecular entities and compositions that do not produce anadverse, allergic or other untoward reaction when administered to ananimal, such as, for example, a human, as appropriate. The preparationof a pharmaceutical composition that contains at least one chimericpolypeptide or additional active ingredient will be known to those ofskill in the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The cell targeted cytotoxic agent, such as serine protease, may comprisedifferent types of carriers depending on whether it is to beadministered in solid, liquid or aerosol form, and whether it need to besterile for such routes of administration as injection. The presenttherapies of the embodiments can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, inhalation (e.g., aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference).

The actual dosage amount of a composition of the present embodimentsadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 5 mg/kg/body weight toabout 100 mg/kg/body weight, about 5 microgram/kg/body weight to about500 milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

In embodiments where compositions are provided in a liquid form, acarrier can be a solvent or dispersion medium comprising but not limitedto, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquidpolyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils,liposomes) and combinations thereof. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin; bythe maintenance of the required particle size by dispersion in carrierssuch as, for example liquid polyol or lipids; by the use of surfactantssuch as, for example hydroxypropylcellulose; or combinations thereofsuch methods. In many cases, it will be preferable to include isotonicagents, such as, for example, sugars, sodium chloride or combinationsthereof.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

VI. Combination Therapies

In order to increase the effectiveness of a nucleic acid, polypeptide ornanoparticle complex of the present embodiments, it may be desirable tocombine these compositions with other agents effective in the treatmentof the disease of interest.

As a non-limiting example, the treatment of cancer may be implementedwith a cell-targeted therapeutic, such as serine protease, of thepresent embodiments along with other anti-cancer agents. An“anti-cancer” agent is capable of negatively affecting cancer in asubject, for example, by killing cancer cells, inducing apoptosis incancer cells, reducing the growth rate of cancer cells, reducing theincidence or number of metastases, reducing tumor size, inhibiting tumorgrowth, reducing the blood supply to a tumor or cancer cells, promotingan immune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. More generally, these other compositions would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cells with theanti-cancer peptide or nanoparticle complex and the agent(s) or multiplefactor(s) at the same time. This may be achieved by contacting the cellwith a single composition or pharmacological formulation that includesboth agents, or by contacting the cell with two distinct compositions orformulations, at the same time, wherein one composition includes theanti-cancer peptide or nanoparticle complex and the other includes thesecond agent(s). In particular embodiments, an anti-cancer peptide canbe one agent, and an anti-cancer nanoparticle complex can be the otheragent.

Treatment with the anti-cancer peptide or nanoparticle-complex mayprecede or follow the other agent treatment by intervals ranging fromminutes to weeks. In embodiments where the other agent and theanti-cancer peptide or nanoparticle complex are applied separately tothe cell, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the agentand the anti-cancer peptide or nanoparticle complex would still be ableto exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one may contact the cell with bothmodalities within about 12-24 hours of each other and, more preferably,within about 6-12 hours of each other. In some situations, it may bedesirable to extend the time period for treatment significantly whereseveral days (e.g., 2, 3, 4, 5, 6 or 7 days) to several weeks (e.g., 1,2, 3, 4, 5, 6, 7 or 8 weeks) lapse between the respectiveadministrations.

Various combinations may be employed, where the targeted cytotoxicagent-based therapy is “A” and the secondary agent, such asradiotherapy, chemotherapy or anti-inflammatory agent, is “B”:

-   -   A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B    -   B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A    -   B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

In certain embodiments, administration of the therapy of the presentembodiments to a patient will follow general protocols for theadministration of chemotherapeutics, taking into account the toxicity,if any, of the vector. It is expected that the treatment cycles would berepeated as necessary. It also is contemplated that various standardtherapies, as well as surgical intervention, may be applied incombination with the described hyperproliferative cell therapy.

A. Chemotherapy

Cancer therapies also include a variety of combination therapies. Insome aspects a serine protease therapeutic of the embodiments isadministered (or formulated) in conjunction with a chemotherapeuticagent. For example, in some aspects the chemotherapeutic agent is aprotein kinase inhibitor such as a EGFR, VEGFR, AKT, Erb1, Erb2, ErbB,Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit,eph receptor or BRAF inhibitors. Nonlimiting examples of protein kinaseinhibitors include Afatinib, Axitinib, Bevacizumab, Bosutinib,Cetuximab, Crizotinib, Dasatinib, Erlotinib, Fostamatinib, Gefitinib,Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Panitumumab,Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib,Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206,GSK690693, A-443654, VQD-002, Miltefosine, Perifosine, CAL101, PX-866,LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, Alvocidib,Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322,ZD1839, P276-00, GW572016 or a mixture thereof.

Yet further combination chemotherapies include, for example, alkylatingagents such as thiotepa and cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall; dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,pteropterin, trimetrexate; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens suchas calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as mitotane, trilostane; folic acidreplenisher such as frolinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes such as cisplatin, oxaliplatin andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum, andpharmaceutically acceptable salts, acids or derivatives of any of theabove. In certain embodiments, the compositions provided herein may beused in combination with gefitinib. In other embodiments, the presentembodiments may be practiced in combination with Gleevac (e.g., fromabout 400 to about 800 mg/day of Gleevac may be administered to apatient). In certain embodiments, one or more chemotherapeutic may beused in combination with the compositions provided herein.

B. Radiotherapy

Radiotherapy has been used extensively in treatments and includes whatare commonly known as γ-rays, X-rays, and/or the directed delivery ofradioisotopes to tumor cells. Other forms radiotherapy are alsocontemplated such as microwaves and UV-irradiation. It is most likelythat all of these factors effect a broad range of damage on DNA, on theprecursors of DNA, on the replication and repair of DNA, and on theassembly and maintenance of chromosomes. Dosage ranges for X-rays rangefrom daily doses of 50 to 200 roentgens for prolonged periods of time (3to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges forradioisotopes vary widely, and depend on the half-life of the isotope,the strength and type of radiation emitted, and the uptake by theneoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic composition and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

C. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually affect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with a serine protease therapy of the present embodiments.The general approach for combined therapy is discussed below. Generally,the tumor cell must bear some marker that is amenable to targeting,i.e., is not present on the majority of other cells. Many tumor markersexist and any of these may be suitable for targeting in the context ofthe present embodiments. Common tumor markers include carcinoembryonicantigen, prostate specific antigen, urinary tumor associated antigen,fetal antigen, tyrosinase (p9′7), gp68, TAG-72, HMFG, Sialyl LewisAntigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb Band p155.

D. Gene Therapy

In yet another embodiment, the secondary treatment is a gene therapy inwhich a therapeutic polynucleotide is administered before, after, or atthe same time as the therapeutic composition. Viral vectors for theexpression of a gene product are well known in the art, and include sucheukaryotic expression systems as adenoviruses, adeno-associated viruses,retroviruses, herpesviruses, lentiviruses, poxviruses including vacciniaviruses, and papiloma viruses, including SV40. Alternatively, theadministration of expression constructs can be accomplished with lipidbased vectors such as liposomes or DOTAP:cholesterol vesicles. All ofthese method are well known in the art (see, e.g. Sambrook et al., 1989;Ausubel et al., 1998; Ausubel, 1996).

Delivery of a vector encoding one of the following gene products willhave a combined anti-hyperproliferative effect on target tissues. Avariety of proteins are encompassed within the present embodiments andare well known in the art.

E. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatmentsprovided herein, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present embodiments may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

VII. Examples

The following examples are included to demonstrate preferred embodimentsof the embodiments. It should be appreciated by those of skill in theart that the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the embodiments, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Granzyme B Construct for Sortase Reaction

Currently, GrB immunotoxins have been made with the GrB already fused tothe targeting moiety which poses substantial production problems. Thus,a GrB cassette was constructed that can be snapped onto the N-terminalend of any of a wide variety of proteins that target tumors using thesortase reaction.

Sortase A is a transpeptidase found in most gram positive bacteria thatcan form new peptide bonds between two proteins one of which containsthe sequence LPXTG(G) (SEQ ID NO: 89) at its C-terminal end and theother of which contains a GGG sequence at it N-terminal end. As shown inFIG. 1, sortase cleaves the Thr-Gly bond in the LPXTG(G) (SEQ ID NO: 89)sequence and forms an intermediate in which it is linked to the Thr viaa thioester bond. The intermediate then reacts with the GGG sequence onthe recipient to form a new peptide bond that links them together(Ton-That et al., 2000; Aulabaugh et al., 2007). The sortase reaction isremarkably specific and, since it takes place under physiologicconditions, it can be used to couple together two recombinant proteinsthat cannot be produced in high yield if already linked as a fusionprotein. It can also be used to couple a drug, or a scaffold that can beloaded with drug, to a recombinant protein (Tsukiji et al., 2009).

To create a form of a serine protease, such as GrB, that can be snappedon to the N-terminal end of any recombinant protein that can be producedwith a GGG sequence at its N-terminal end, a construct was achieved bymodifying the structure of GrB to include two flexible spacers followedby the LPETGG sequence that allows the sortase reaction to form apeptide bond between the spacers at the C-terminal end of GrB and theN-terminal end of a tumor targeting protein. A GrB-(G₄S)₂-LPETGGexpressing vector was designed to have a 6×His tag on the N-terminal endfor purification followed by an EK cut site that allows removal of the6×His tag in the final product (FIG. 2). The vector was transfected intoCHO-S cells, selected in zeocin under serum-free conditions for threeweeks and then dilutionally cloned at 0.5 cell/well into 16 96-wellplates. After single clones were grown and expanded, the expressionlevels of the recombinant GrB-(G₄S)₂-LPETGG clones were assessed byWestern blot analysis using anti-GrB antibody (FIG. 3). 17 positiveclones were identified out of total 112 clones screened with Clone #31producing the highest level of GrB-(G₄S)₂-LPETGG.

Example 2—Characterization of GrB-(G₄S)₂-LPETGG Construct

To facilitate a detailed study of the ability of the GrB-(G₄S)₂-LPETGGand other warheads to form a peptide bond in the sortase reaction, twotagged peptides that have been reported to function well as acceptors inthe sortase reaction (FIG. 4) were synthesized. The peptide shown inFIG. 4A (GGG-biotin) contains a biotin tag so that the product of thesortase reaction can be captured by streptavadin and its amountquantified by Western blot analysis. The peptide shown in FIG. 5B(GGG-fluorochrome) contains a fluorochrome that can be visualizeddirectly. In order to allow the use of a Licor Odyssey instrument forprecise quantification, the fluorochrome can be changed from Alexa₆₄₇ toAlexa₇₀₀ or Alexa₇₂₀ depending on solubility. These peptides, incombination with the 6×His tag on the N-terminal end of the GrB willallow the determination of the kinetics and completeness of the sortasereaction in detail.

Having established that the novel GrB warhead functions in the sortasereaction, a targeting moiety was attached in the form of yoked humanchorionic gonadotropin (YCG) in which the α and β chains of the hCGhormone have been fused together to form a single protein as reportedpreviously (Kanatani et al, 2011). The YCG was molecularly modified toadd a triglycine sequence (GGG) followed by either 1 or 2 G₄S spacers tothe N-terminal end of YCG so that it can function as an acceptor in thesortase reaction. These experiments established the principle that GrBwarhead can be snapped onto another protein and will lead to furtherexploration of this reaction by attempting to snap GrB onto additionaltumor targeting systems including those based on dendrimer folates.

Example 3—Use of Sortase Reaction to Create a Molecule Consisting of YCGLinked to MMAE

The results of Example 2 and studies using with GrB-YCG produced in Sf9cells confirmed earlier studies suggesting that the LHR is selectivelyexpressed on reproductive system tumors, and that it can be targetedeffectively with YCG. Thus, there is a strong rationale for attemptingto arm it with several different types of warheads. The extremely potentcytotoxic monomethylaurostatin E (MMAE) is being widely used to armantibodies by linking it through a citrulline-valine protease-cleavablelinker. It was hypothesized that the sortase reaction can be used to armYCG by attaching a single molecule of valine-citrulline-MMAE (vc-MMAE)to the C-terminal end (FIG. 7).

The sortase reaction required an LPETGG sequence at the C-terminal endof the donor molecule and a GGG sequence at the N-terminal end of theacceptor molecule. The MMAE had a triglycine sequence and acitrulline-valine cleavable linker added[(glycine)₃-valine-citruline-MMAE, abbreviated (GGG-vc-MMAE) and sortasewas produced as a recombinant protein in E. coli. Previous attempts tolink YCG-LPETGG produced in Sf9 and CHO cells to chemically synthesizedGGG-vc-MMAE in the presence of sortase were unable to obtain the finalYCG-vc-MMAE product. It was suspected that access of the sortase to theLPETGG sequence at the C terminus of YCG may have been stericallyhindered. Thus, a flexible spacer was added between the C-terminus andthe LPETGG sequence. The molecule was re-engineered to put two (G₄S)spacers between YCG and LPETGG. Simultaneously, based on the success inproducing other proteins at higher yield in CHO cells, YCG-(G₄S)₂-LPETGGwas cloned into the pSecTag vector and transfected in CHO-S cells forselection of YCG-(G₄S)₂-LPETGG expressing stable clones. TwentyYCG-(G₄S)₂-LPETGG expressing clones were identified out of 75 clonesscreened and Clone #22 was chosen for large-scale expression andsubsequent experiments since it produces the highest level and cleanestYCG-(G₄S)₂-LPETGG (FIG. 8).

Following IMAC purification, it was next determined whetherYCG-(G₄S)₂-LPETGG would work in the sortase reaction by using G5K-biotinas the acceptor. As shown in FIG. 9, the reaction ran well and theamount of product increased with the concentration of G5K-biotin.

The sortase reaction of YCG-(G₄S)₂-LPETGG with GGG-vc-MMAE was carriedout at the ratio of 1:1:5 (sortase:donor:acceptor) at 37° C. for 6 h. Asshown in FIG. 10, anti-MMAE antibody was able to detect a signal at a MWclose to YCG-(G₄S)₂-LPETGG in the reaction mixture of sortase (lane 4)indicating that MMAE was successfully ligated to YCG-(G₄S)₂-LPETGGyielding the YCG-MMAE final product, which was also confirmed byblotting with anti-hCG antibody. The YCG-MMAE product is furtherpurified using a combination of IMAC and dialysis, and its cytotoxicityis tested in vitro against isogenic LHR^(WT) and LHR^(KD) cells whichexpress high levels of the LH receptor and a subline in which expressionhas been knocked down.

Example 4—Use of R-Spondins to Target Cytotoxin to LGR6-Expressing TumorStem Cells

The pMAL-p5X vector was used to construct vectors that express eitherR-spondin 1 or R-spondin 2 connected through a pair of GGGGS (SEQ ID NO:36) linkers to the LPETGG sequence (RSPO1-(G₄S)₂-LPETGG andRSPO2-(G₄S)₂-LPETGG). These were designed to have a MBP tag and EK cutsite on the N-terminal end and 6×His tag on the C-terminal end forinitial purification and final isolation of the end product (FIG. 11).The recombinant RSPO1-(G₄S)₂-LPETGG and RSPO2-(G₄S)₂-LPETGG proteinswere effectively expressed as a soluble MBP fusion proteins in E. coli,and after purification by Ni-NTA metal-affinity chromatography (IMAC)they had the expected molecular masses of 64 kDa and 65 kDa,respectively.

Experiments were conducted to determine whether eitherRSPO1-(G₄S)₂-LPETGG or RSPO2-(G₄S)₂-LPETGG would serve as a donor in thesortase reaction. As shown in FIG. 12, using a small bioreactor,GGG-vc-MMAE was effectively coupled to both of these proteins in aconcentration-dependent manner.

Two approaches were used to establish isogenic test systems forassessment of LGR6-dependent selectivity of killing by RSPO-MMAE. LGR6was overexpressed in cells that have a low level of this protein andboth alleles of LGR6 were knocked down in an ovarian cancer cell linethat expresses high levels of LGR6. Both HEK293 embryonal cells andKuramochi ovarian cancer cells were transfected with a plasmidexpressing the LGR6 cDNA and stable, high-expressing populations wereisolated by Western blot screening. As shown in FIG. 3, a population ofHEK293 cells that express a much higher level of LGR6 than the parentalHEK293 cells was isolated.

Empty vector-transfected HEK293 cells and population #2 of the LGR6over-expressing HEK293 cells were used to test sensitivity to the“MBP-RSPO1-MMAE”. As shown in FIG. 14, MBP-RSPO1-MMAE is 12-fold morepotent at killing LGR6-overexpressing HEK-LGR6-2 cells than emptyvector-transfected HEK-pcDNA cells (IC₅₀ 0.08 nM versus 0.95 nM). Thus,the sortase reaction can be used to develop targeted therapeutic agents.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A cell-targeted cytotoxic construct comprising atargeting moiety that binds to tumor stem cells and a cytotoxic moiety,the construct comprising: (a) an R-spondin-1 (RSPO1) targetingpolypeptide ligand that comprises two-tandem furin-like cysteine-rich(Fu-CRD) domains of RSPO1, wherein the targeting ligand binds to a cellsurface human LGR 5 and LGR 6 receptors; (b) a linking polypeptide thatcomprises LPETG (SEQ ID NO: 66), the polypeptide being positionedbetween the targeting ligand and a cytotoxic moiety; and (c) a cytotoxicmoiety that is MMAE; wherein the RSPO1 is bound to the N-terminus of thelinking polypeptide and the MMAE is bound to the C-terminus of thelinking polypeptide.
 2. The cell-targeted construct of claim 1, whereinthe linking polypeptide comprises LPETGG.
 3. The cell-targeted constructof claim 2, wherein the construct further comprises at least one spacerpositioned between the RSPO1 and the polypeptide linker.
 4. The compoundof claim 3, wherein the construct comprises two spacers.
 5. Theconstruct of claim 3, wherein the spacer comprises the sequence G₄S(GGGGS; SEQ ID NO: 36).